Phylogenetic analyses of Saussurea sect. Pseudoeriocoryne (Asteraceae : Cardueae) based on chloroplast DNA trnL-F sequences

The genus Saussurea is distributed mainly in the temperate and subarctic regions of Eurasia and consists of about 300 species classified into six subgenera and 20 sections. Sect. Pseudoeriocoryne in the subgenus Eriocoryne comprises four species, and is delimited mainly by acaulescence and an inflorescence with congested capitula surrounded by a rosette of leaves. All of these species are endemic to the and Qinghai-Tibet Plateau. Sequences from the chloroplast DNA trnL-F region were obtained for the four species in this section and 26 other species from four subgenera of Saussurea to resolve phylogenetic relationships among these species and to determine whether the shared characters that define sect. Pseudoeriocoryne are synapomorphic or were acquired by convergent evolution. The resulting phylogenies indicated that Saussurea sect. Pseudoeriocoryne as traditionally defined does not constitute a monophyletic group and that each of its species belongs to separate clades. Furthermore, none of these species showed a close relationship with the other species of subgenus Eriocoryne. Our results further indicated that none of the investigated subgenera are monophyletic, and that species from different subgenera clustered together. All these conclusions are provisional and their confirmation would require stronger phylogenetic support. Two possible explanations are suggested for low sequence divergence, poor resolution of internal clades and clustering of species with the rather distinct morphology of Saussurea detected in the present study. The first is rapid radiation and diversification triggered by fast habitat fragmentation due to the recent lifting of the Qinghai-Tibet Plateau and the Quaternary climate oscillations. This could have led to rapid morphological divergence while sequences diverged very little, and also caused the convergent acquisition of similar characteristics in unrelated lineages due to similar selection pressures. The second possible explanation is that both introgressive hybridization and reticulate evolution might have caused the transferring of cpDNA sequences between morphologically dissimilar species, thus leading to homogenization of sequences between lineages. (C) 2004 Elsevier Ltd. All rights reserved.

Burrowing rodents as ecosystem engineers: the ecology and management of plateau zokors Myospalax fontanierii in alpine meadow ecosystems on the Tibetan Plateau

1. Plateau zokors, Myospalax fontanierii, are the only subterranean herbivores on the Tibetan plateau of China. Although the population biology of plateau zokors has been studied for many years, the interactions between zokors and plants, especially for the maintenance and structure of ecological communities, have been poorly recognized. In the past, plateau zokors have been traditionally viewed as pests, competitors with cattle, and agents of soil erosion, thus eradication programmes have been carried out by local governments and farmers. Zokors are also widely and heavily exploited for their use in traditional Chinese medicine.2. Like other fossorial animals, such as pocket gophers Geomys spp. and prairie dogs Cynomys spp. in similar ecosystems, zokors may act to increase local environmental heterogeneity at the landscape level, aid in the formation, aeration and mixing of soil, and enhance infiltration of water into the soil thus curtailing erosion. The changes that zokors cause in the physical environment, vegetation and soil clearly affect the herbivore food web. Equally, plateau zokors also provide a significant food source for many avian and mammalian predators on the plateau. Zokor control leading to depletion of prey and secondary poisoning may therefore present problems for populations of numerous other animals.3. We highlight the important role plateau zokors play in the Tibetan plateau ecosystem. Plateau zokors should be managed in concert with other comprehensive rangeland treatments to ensure the ecological equilibrium and preservation of native biodiversity, as well as the long-term sustainable use of pastureland by domestic livestock.

Molecular evidence for the sister relationship of the eastern Asia-North American intercontinental species pair in the Podophyllum group (Berberidaceae)

The presumed pair relationships of intercontinental vicariad species in the Podophyllum group (Sinopodophyllum hexandrum vs. Podophyllum pelatum and Diphylleia grayi vs. D. cymosa) were recently, considered to be paraphyletic. In the present paper, the trnL-F and ITS gene sequences of the representatives were used to examine the sister relationships of these two vicariad species. A heuristic parsimony analysis based on the trnLF data identified Diphylleia as the basal clade of the other three genera, but provided poor resolution of their inter-relationships. High sequence divergence was found in the ITS data. ITS1 region, more variable but parsimonyuninformative. has no phylogenetic value, Sequence divergence of the ITS2 region provided abundant, phylogenetically informative variable characters. Analysis of ITS2 sequences confirmeda sister relationship between the presumable vicariad species, in spite of a low bootstrap support for Sinopodophyllum hexandrum vs. Podophyllum pelatum. The combined ITS2 and trnL-F data enforced a sister relationship between Sinopodophyllum hexandrum and Podophyllum pelatum with an elevated bootstrap support of 100%. Based on molecular phylogeny, the morphological evolution of this group was discussed. The self-pollination might have evolved from cross-fertilization two times in this group. The different pollination and seed dispersal systems of Sinopodophyllum hexandrum and Podophlyllum pelatum resulted from their adaptations to different ecological habitats. The divergence time of Sinopodophyllum hexandrum-Podophyllum pelatum is estimated to be 6.52+/-1.89 myr based on the ITS divergence. The divergence of this species pair predated or co-occurred with the recent uplift of the Himalayas 4-3 myr during the late Miocene and the formation of the alpine habitats. Sinopodophyllum hexandrum developed a host of specialized characters in its subsequent adaptation to the arid alpine surroundings. The present study confirmed the different patterns of species relationship between Asian-North American disjuncts. The isolation of plant elements between North America and eastern Asia must have been a gradual process, resulting in the different phylogenetic patterns and divergence times of the disjuncts.

Phylogenetic relationships of five pika species (genus Ochotona) based on mitochondrial DNA restriction maps

Restriction site mapping of mitochondrial DNA (mtDNA) with 16 restriction endonucleases was used to examine the phylogenetic relationships of Ochotona cansus, O. huangensis, O. thibetana, O. curzoniae and O. erythrotis. A 1-kb length variation between O. erythrotis of subgenus Pika and other four species of subgenus Ochotona was observed, which may be a useful genetic marker for identifying the two subgenera. The phylogenetic tree constructed using PAUP based on 61 phylogenetically informative sites suggests that O. erythrotis diverged first, followed by O. cansus, while O. curzoniae and O. huangensis are sister taxa related to O. thibetana, The results indicate that both O. cansus and O. huangensis should be treated as independent species. If the base substitution rate of pikas mtDNA was 2% per million years, then the divergence time of the two subgenera, Pika and Ochotana, is about 8.8 Ma ago of late Miocence, middle Bao-dian of Chinese mammalian age, and the divergence of the four species in subgenus Ochotona would have occurred about 2.5 - 4.2 Ma ago, Yushean of Chinese mammalian age. This calculation appears to be substantiated by the fossil record.

甘肃临夏盆地龙担剖面晚新生代磁性地层与古环境

Linxia Basin, situated in the northeast belt of the Tibetan Plateau, is a late Cenozoic depression basin bounded by the Tibetan Plateau and the Chinese Loess Plateau. The Cenozoic deposition, spanning over 30Ma, in which very abundant mammal fossils were discovered, is very suitable for study of uplift processes and geo-morphological evolution of the Tibetan Plateau. The Longdan section (35°31′31.6″N，103°29′0.6″E) is famous for the middle Miocene Platybelodon fauna and the late Miocene Hipparion fauna for a long time and is also one of the earliest known places for wooly rhino, which lies on the east slope of Longdan, a small village of township Nalesi in the south of the Dongxiang Autonomous County, Linxia Hui Nationallity Autonomous Prefecture. The Longdan mammal fauna was discovered at the base of the Early Pleistocene loess deposits at Dongxiang, where the lithology is different from the typical Wucheng Loess on the Chinese Loess Plateau. The rich fossils contain many new species and the major two layers of fossils are in the loess beds. Geologically the fossiliferous area is located in the central part of the Linxia Cenozoic sedimentary basin. Tectonically the Linxia Basin is an intermountain fault basin, bordered by the Leijishan major fault in the south and the north Qinling and Qilianshan major faults in the north. The section is 51.6m thick above the gravel layer, including the 1.6m Late Pleistocene Malan Loess on the top and the other loess-paleosol sequences in the middle of the section. The base of the section is the Jishi Formation, consisting of gravel layer of 13 ~ 17m thick. In this study, 972 bulk samples were collected with an interval of 5cm and other 401 orientied samples were taken with a magnetic compass. In the laboratory, the paleomagnetism, medium grain size, susceptibility, color, micromorphology, anisotropy of magnetic susceptibility were analyzed. From the stratigraphic analysis, the Longdan section from the top 0.3m to the bottom 51.6m, containing 5 normal polarities (N1-N5) and 5 reversal polarities (R1-R5). The paleomagnetic results show N3 is the Olduvai subchron in the middle of the Matuyama chron, and then the chronology of the Longdan mammal fauna is constructed along the section. The Matuyama-Gauss boundary is 45m and N5 enters Gauss chron. The Olduvai subchron with the age of 1.77 ~ 1.95Ma is found just in the upper fossiliferous level of Longdan mammal fauna. Taking the deposit rate of the section into account, the geological age of the upper fossiliferous level of Longdan mammal fauna is estimated to be about 1.9Ma. The lower fossiliferous level is just below the Reunion subchron and its age is estimated to be 2.25Ma. In addition, anisotropy of magnetic susceptibility of the loess-paleosol and other climatic indexes were used for discussing the late Cenozoic paleoenvironmental changes at Longdan, from which the Longdan area should have been an area of predominantly steppe the same as the Longdan mammal fauna.

Fusion Artmap: A Neural Network Architecture for Multi-Channel Data Fusion and Classification

Fusion ARTMAP is a self-organizing neural network architecture for multi-channel, or multi-sensor, data fusion. Single-channel Fusion ARTMAP is functionally equivalent to Fuzzy ART during unsupervised learning and to Fuzzy ARTMAP during supervised learning. The network has a symmetric organization such that each channel can be dynamically configured to serve as either a data input or a teaching input to the system. An ART module forms a compressed recognition code within each channel. These codes, in turn, become inputs to a single ART system that organizes the global recognition code. When a predictive error occurs, a process called paraellel match tracking simultaneously raises vigilances in multiple ART modules until reset is triggered in one of them. Parallel match tracking hereby resets only that portion of the recognition code with the poorest match, or minimum predictive confidence. This internally controlled selective reset process is a type of credit assignment that creates a parsimoniously connected learned network. Fusion ARTMAP's multi-channel coding is illustrated by simulations of the Quadruped Mammal database.

Acoustic behaviour, ecology and social structure of bottlenose dolphins (Tursiops truncatus, Montagu 1821) in the North Atlantic

Communication is important for social and other behavioural interactions in most marine mammal species. The bottlenose dolphin (Tursiops truncatus, Montagu, 1821) is a highly social species that use whistles as communication calls to express identity and to initiate and maintain contact between socially interactive individuals. In this thesis, the degree of variability in whistle behaviour and whistle characteristics was examined between different habitats on a range of spatial scales. The whistle characteristics that best discriminated between different communities were investigated, along with exploration of whistle variation in relation to habitat type, levels of social interaction and relatedness. Finally, the use and variability of individually distinctive calls (signature whistles) within and between Irish and US waters were also examined. Relatively high levels of whistle variation were found within a genetically and socially isolated population of dolphins in the Shannon Estuary, reflecting the need for individual identification and distinctive whistles in a population with long term site fidelity and high levels of social cohesion. Variation between reproductively separate communities in Irish waters was relatively small except between animals in inshore compared with continental shelf waters. The greatest differences in whistle structure overall were evident between dolphins using inshore and offshore US waters, likely reflecting social isolation of the two distinct ecotypes that occur in these waters but also variation in behaviour or habitat conditions. Variation found among inshore communities in US waters reflected similarities in habitat use and levels of social interaction. These findings suggest that vocal variation is socially mediated, behaviourally maintained and dependent on levels of social contact between individuals. The findings contribute to our understanding of the interaction of factors influencing vocalisation behaviour in this behaviourally complex and ecologically plastic species.

Functional genomics of commensal Lactobacilli

Catabolic flexibility affords a bacterium the ability to utilise different sugar sources as carbon for energy. This is important for commensal lactobacilli like Lactobacillus ruminis which can be exposed to a variety of carbohydrates in vivo. However, little is known about the fermentation capabilities, metabolic pathways, genetic diversity or potential survival mechanisms used by L. ruminis in vivo. A combination of in vitro and in silico techniques was used to identify the catabolic pathways of L. ruminis. I also compared 16 L. ruminis strains using a panel of biochemical and survival assays, genetically, whole genome sequencing and RNA sequencing. Multi locus sequence typing revealed that strains clustered according to their host sources. Transcriptome analysis by RNAseq of two motile strains under three growth conditions, including swarming, identified the up-regulation of carbohydrate-related genes under swarming conditions. This suggests that carbohydrate flexibility may have an uncharacterised role in L. ruminis swarming. Following on from the assessment of L. ruminis catabolic flexibility, the porcine diet was supplemented with galactooligosaccharides or L. ruminis ATCC 25644 plus galactooligosaccharides. Supplementation of the porcine diet with galactooligosaccharide had no effect on microbiota diversity. In contrast, the L. ruminis plus galactooligosaccharide treatment significantly reduced the microbiota diversity. Diet is a major factor that affects the diversity of the gut microbiota. In order to get a more thorough understanding of diet and gut health in animals such as racehorses and domesticated herbivores, I determined the core microbiota of animals consuming different feeds. Interestingly, the gut microbiota diversity correlated with the host phylogeny of the animal. The genome of Lactobacillus equi (2.19 Mb), isolated from a healthy Irish thoroughbred was also sequenced and annotated, and comprised 2,263 predicted genes. The large repertoire of predicted carbohydrate-related genes may offer L. equi an advantage in the complex and harsh hindgut environment. In summary, this thesis uses functional genomics to assess the effect that carbohydrates have on commensal lactobacilli and the microbiota as a whole.

Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

Trophic role of small cetaceans and seals in Irish waters

Understanding the role of marine mammals in specific ecosystems and their interactions with fisheries involves, inter alia, an understanding of their diet and dietary requirements. In this thesis, the foraging ecology of seven marine mammal species that regularly occur in Irish waters was investigated by reconstructing diet using hard parts from digestive tracts and scats. Of the species examined, two (striped and Atlantic white-sided dolphin) can be considered offshore species or species inhabiting neritic waters, while five others usually inhabit more coastal areas (white-beaked dolphin, harbour porpoise, harbour seal and grey seal); the last species studied was the bottlenose dolphin whose population structure is more complex, with coastal and offshore populations. A total of 13,028 prey items from at least 81 different species (62 fish species, 14 cephalopods, four crustaceans, and a tunicate) were identified. 28% of the fish species were identified using bones other than otoliths, highlighting the importance of using all identifiable structures to reconstruct diet. Individually, each species of marine mammal presented a high diversity of prey taxa, but the locally abundant Trisopterus spp. were found to be the most important prey item for all species, indicating that Trisopterus spp. is probably a key species in understanding the role of these predators in Irish waters. In the coastal marine mammals, other Gadiformes species (haddock, pollack, saithe, whiting) also contributed substantially to the diet; in contrast, in pelagic or less coastal marine mammals, prey was largely comprised of planktivorous fish, such as Atlantic mackerel, horse mackerel, blue whiting, and mesopelagic prey. Striped dolphins and Atlantic white-sided dolphins are offshore small cetaceans foraging in neritic waters. Differences between the diet of striped dolphins collected in drift nets targeting tuna and stranded on Irish coasts showed a complex foraging behaviour; the diet information shows that although this dolphin forages mainly in oceanic waters it may occasionally forage on the continental shelf, feeding on available prey. The Atlantic white-sided dolphin diet showed that this species prefers to feed over the continental edge, where planktivorous fish are abundant. Some resource partitioning was found in bottlenose dolphins in Irish waters consistent with previous genetic and stable isotope analysis studies. Bottlenose dolphins in Irish waters appears to be generalist feeders consuming more than 30 prey species, however most of the diet comprised a few locally abundant species, especially gadoid fish including haddock/pollack/saithe group and Trisopterus spp., but the contribution of Atlantic hake, conger eels and the pelagic planktivorous horse mackerel were also important. Stomach content information suggests that three different feeding behaviours might occur in bottlenose dolphin populations in Irish waters; firstly a coastal behaviour, with animals feeding on prey that mainly inhabit areas close to the coast; secondly an offshore behaviour where dolphins feed on offshore species such as squid or mesopelagic fish; and a third more complex behaviour that involves movements over the continental shelf and close to the shelf edge. The other three coastal marine mammal species (harbour porpoise, harbour seal and grey seal) were found to be feeding on similar prey and competition for food resources among these sympatric species might occur. Both species of seals were found to have a high overlap (more than 80%) in their diet composition, but while grey seals feed on large fish (>110mm), harbour seals feed mostly on smaller fish (<110mm), suggesting some spatial segregation in foraging. Harbour porpoises and grey seals are potentially competing for the same food resource but some differences in prey species were found and some habitat partitioning might occur. Direct interaction (by catch) between dolphins and fisheries was detected in all species. Most of the prey found in the stomach contents from both stranded and by catch dolphins were smaller sizes than those targeted by commercial fisheries. In fact, the total annual food consumption of the species studied was found to be very small (225,160 tonnes) in comparison to fishery landings for the same area (~2 million tonnes). However, marine mammal species might be indirectly interacting with fisheries, removing forage fish. Incorporating the dietary information obtained from the four coastal species, an ECOPATH food web model was established for the Irish Sea, based on data from 2004. Five trophic levels were found, with bottlenose dolphins and grey and harbour seals occurring at the highest trophic level. A comparison with a previous model based on 1973 data suggests that while the overall Irish Sea ecosystem appears to be “maturing”, some indices indicate that the 2004 fishery was less efficient and was targeting fish at higher trophic levels than in 1973, which is reflected in the mean trophic level of the catch. Depletion or substantial decrease of some of the Irish Sea fish stocks has resulted in a significant decline in landings in this area. The integration of diet information in mass-balance models to construct ecosystem food-webs will help to understand the trophic role of these apex predators within the ecosystem.

Newly resolved relationships in an early land plant lineage: Bryophyta class Sphagnopsida (peat mosses).

UNLABELLED: PREMISE OF THE STUDY: The Sphagnopsida, an early-diverging lineage of mosses (phylum Bryophyta), are morphologically and ecologically unique and have profound impacts on global climate. The Sphagnopsida are currently classified in two genera, Sphagnum (peat mosses) with some 350-500 species and Ambuchanania with one species. An analysis of phylogenetic relationships among species and genera in the Sphagnopsida were conducted to resolve major lineages and relationships among species within the Sphagnopsida. • METHODS: Phylogenetic analyses of nucleotide sequences from the nuclear, plastid, and mitochondrial genomes (11 704 nucleotides total) were conducted and analyzed using maximum likelihood and Bayesian inference employing seven different substitution models of varying complexity. • KEY RESULTS: Phylogenetic analyses resolved three lineages within the Sphagnopsida: (1) Sphagnum sericeum, (2) S. inretortum plus Ambuchanania leucobryoides, and (3) all remaining species of Sphagnum. Sister group relationships among these three clades could not be resolved, but the phylogenetic results indicate that the highly divergent morphology of A. leucobryoides is derived within the Sphagnopsida rather than plesiomorphic. A new classification is proposed for class Sphagnopsida, with one order (Sphagnales), three families, and four genera. • CONCLUSIONS: The Sphagnopsida are an old lineage within the phylum Bryophyta, but the extant species of Sphagnum represent a relatively recent radiation. It is likely that additional species critical to understanding the evolution of peat mosses await discovery, especially in the southern hemisphere.

Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler.

BACKGROUND: The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage. RESULTS: We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli. CONCLUSION: The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Unprecedented loss of ammonia assimilation capability in a urease-encoding bacterial mutualist.

BACKGROUND: Blochmannia are obligately intracellular bacterial mutualists of ants of the tribe Camponotini. Blochmannia perform key nutritional functions for the host, including synthesis of several essential amino acids. We used Illumina technology to sequence the genome of Blochmannia associated with Camponotus vafer. RESULTS: Although Blochmannia vafer retains many nutritional functions, it is missing glutamine synthetase (glnA), a component of the nitrogen recycling pathway encoded by the previously sequenced B. floridanus and B. pennsylvanicus. With the exception of Ureaplasma, B. vafer is the only sequenced bacterium to date that encodes urease but lacks the ability to assimilate ammonia into glutamine or glutamate. Loss of glnA occurred in a deletion hotspot near the putative replication origin. Overall, compared to the likely gene set of their common ancestor, 31 genes are missing or eroded in B. vafer, compared to 28 in B. floridanus and four in B. pennsylvanicus. Three genes (queA, visC and yggS) show convergent loss or erosion, suggesting relaxed selection for their functions. Eight B. vafer genes contain frameshifts in homopolymeric tracts that may be corrected by transcriptional slippage. Two of these encode DNA replication proteins: dnaX, which we infer is also frameshifted in B. floridanus, and dnaG. CONCLUSIONS: Comparing the B. vafer genome with B. pennsylvanicus and B. floridanus refines the core genes shared within the mutualist group, thereby clarifying functions required across ant host species. This third genome also allows us to track gene loss and erosion in a phylogenetic context to more fully understand processes of genome reduction.

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.