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Avian embryo susceptibility to Italian H7N1 avian influenza viruses belonging to different lineages.

An immunohistochemical investigation was performed to assess tissue tropism and viral replication of Italian H7N1 isolates belonging to different lineages in developing chicken, turkey, Muscovy duck, and mallard duck embryos. Low-pathogenic avian influenza (LPAI)isolates were selected on the basis of the location in the phylogenetic tree; a progenitor strain, A/turkey/Italy/977/V99 (exhibiting no additional glycosylation sites, nAGS), strain A/turkey/Italy/2379/V99 (AGS in position 123), and strain A/turkey/Italy/3675/V99 (AGS in position 149) were selected. The latter two strains belonged to distinct lineages originating from the pool of progenitor strains. The highly pathogenic avian influenza (HPAI) isolate A/turkey/Italy/4580/V99 was also included in the test. All the embryos tested supported the growth of HPAI. The LPAI isolates replicated readily in the allantoic layer of the chorioallantoic membrane of all the species tested and did not replicate to detectable levels in the developing chicken, turkey, and Muscovy duck embryos. In contrast, they replicated to different extents in the respiratory tract of the developing mallard embryo. The findings indicate that the pathogenesis of LPAI infections in mallard embryos is different to that observed in other species and should be investigated further.

Animals↗

Rate heterogeneity among lineages of tracheophytes: integration of molecular and fossil data and evidence for molecular living fossils.

Many efforts to date evolutionary divergences by using a molecular clock have yielded age estimates that are grossly inconsistent with the paleontological evidence. Such discrepancies often are attributed to the inadequacy of the fossil record, but many potential sources of error can affect molecular-based estimates. In this study, we minimize the potential error caused by inaccurate topology and uncertain calibration times by using a well-supported tree, multiple genes, and multiple well-substantiated dates to explore the correspondence between the fossil record and molecular-based age estimates for major clades of tracheophytes. Age estimates varied because of gene effects, codon position, lineage effects, method of inferring branch lengths, and whether or not rate constancy was assumed. However, even methods designed to ameliorate the effects of rate heterogeneity among lineages could not accommodate the substantially slower rates observed in Marattia + Angiopteris and in the tree ferns. Both of these clades of ferns have undergone dramatic decelerations in their rates of molecular evolution and are "molecular living fossils," consistent with their relative morphological stasis for the past 165-200 million years. Similar discrepancies between the fossil record and molecular-based age estimates noted in other studies may also be explained in part by violations of rate constancy among lineages.

Journal Article↗

Evolutionary pattern of the H 3 haemagglutinin of equine influenza viruses: multiple evolutionary lineages and frozen replication.

The nucleotide and deduced amino acid sequences of the haemagglutinin genes coding for the HA 1 domain of H3N8 equine influenza viruses isolated over wide regions of the world were analyzed in detail to determine their evolutionary relationships. We have constructed a phylogenetic model tree by the neighbour-joining method using nucleotide sequences of 15 haemagglutinin genes, including those of five viruses determined in the present study. This gene tree revealed the existence of two major evolutionary pathways during a twenty five-year period between 1963 to 1988, and each pathway appeared to consist of two distinct lineages of haemagglutinin genes. Furthermore, our analysis of nucleotide sequences showed that two distinct lineages of equine H3N8 viruses were involved in an equine influenza outbreak during the period of December 1971-January 1972 in Japan. The number of nucleotide changes between strains was proportional to the length of time (in years) between their isolation except for three of the HA genes. However, there are three exceptional strains isolated in 1971, 1987, and 1988, respectively. The haemagglutinin gene in these strains showed a small number of nucleotide substitutions after they branched off around 1963, suggesting an example of frozen replication. Although the estimated rate (0.0094/site/year) of synonymous (silent) substitutions of the haemagglutinin gene of equine H3N8 viruses was nearly the same as that of human H 1 and H 3 haemagglutinin genes, the rate of nonsynonymous (amino-acid changing) substitutions of the former equine virus gene was estimated to be 0.00041/site/year--that is about 5 times lower than that estimated for the human H 3 haemagglutinin gene. The present study is the first demonstration that multiple evolutionary lineages of equine H3N8 influenza virus circulated since 1963.

Amino Acid Sequence↗

Genetic diversity, reproductive biology, and speciation in the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin.

Beauveria bassiana, a mitosporic fungus used for the biological control of many insect species, is recognized as a "species complex" comprising genetically diverse lineages. Being predominantly asexual, mating tests cannot be applied to delimit species in this species complex. Genetic tests offer an indirect means of identifying species among isolates. To this end, molecular genetic analysis of a sample of B. bassiana isolates with 2 subsamples, 1 representing a worldwide collection and another from a localized epizootic population was carried out. DNA markers generated through AFLPs (amplified fragment length polymorphisms) and SSCPs (single-strand conformation poly morphisms) and nucleotide sequence data of different allelic forms of 3 genes (large and small subunits of rRNA and beta-tubulin) were evaluated. The B. bassiana isolates from the worldwide sample showed 11% overall similarity and no closely clustered groups. Phylogenetic trees generated from the AFLP and SSCP data of this sample resolved the different isolates into distinct phylogenetic lineages. In the epizootic B. bassiana population, prevalence of recombination was evident from random association of alleles in multilocus tests and lack of phylogenetic concordance among 3 gene genealogies. Thus, the worldwide sample of B. bassiana exhibits a predominantly clonal structure, hinting at species divergence leading to cryptic speciation with recombination being customary among isolates sharing a close ecological niche.

Animals↗

Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees.

In the mycorrhizal symbiosis, plants exchange photosynthates for mineral nutrients acquired by fungi from the soil. This mutualistic arrangement has been subverted by hundreds of mycorrhizal plant species that lack the ability to photosynthesize. The most numerous examples of this behaviour are found in the largest plant family, the Orchidaceae. Although these non-photosynthetic orchid species are known to be highly specialized exploiters of the ectomycorrhizal symbiosis, photosynthetic orchids are thought to use free-living saprophytic, or pathogenic, fungal lineages. However, we present evidence that putatively photosynthetic orchids from five species which grow in the understorey of forests: (i) form mycorrhizas with ectomycorrhizal fungi of forest trees; and (ii) have stable isotope signatures indicating distinctive pathways for nitrogen and carbon acquisition approaching those of non-photosynthetic orchids that associate with ectomycorrhizal fungi of forest trees. These findings represent a major shift in our understanding of both orchid ecology and evolution because they explain how orchids can thrive in low-irradiance niches and they show that a shift to exploiting ectomycorrhizal fungi precedes viable losses of photosynthetic ability in orchid lineages.

Analysis of Variance↗

Can microsatellites be used to infer phylogenies? Evidence from population affinities of the Western Canary Island lizard (Gallotia galloti).

Population phylogeographic studies are generally based solely on mtDNA without corroboration, from an independent segregating unit (i.e., nuclear genes), that the mtDNA gene tree represents the organismal phylogeny. This paper attempts to evaluate the utility of microsatellites for this process by use of the Western Canary Island lacertid (Gallotia galloti) as a model. The geological times of island eruptions are known, and well-supported mtDNA phylogenies exist (corroborated as the organismal phylogeny rather than just a gene tree by nuclear random amplified polymorphic DNAs (RAPDs)). The allelic variation in 12 populations from four islands (representing five haplotype lineages) was investigated in five unlinked microsatellite loci. Analysis of molecular variance showed this data to be highly structured. A series of genetic distances among populations was computed based on both the variance in allele frequency (i.e., F(st) related) and the variance in repeat numbers (i.e., R(st) related). The genetic distances based on the former were more highly correlated with the mtDNA genetic distances than those based on the latter. All trees based on both models supported the primary division shown by mtDNA and RAPDs, which is dated at ca. 2.8 to 5.6 mybp (depending on calibration of the mtDNA clock) and which could, under the evolutionary species concept, be regarded separate species. This was achieved despite theoretical problems posed by the use of few loci, suspected bottlenecks, and large population sizes. The finer details were less consistently represented. Nevertheless, this study demonstrates that even a small number of microsatellites can be useful in corroborating the deeper divisions of a population phylogeny.

Alleles↗

A likelihood framework for inferring the evolution of geographic range on phylogenetic trees.

At a time when historical biogeography appears to be again expanding its scope after a period of focusing primarily on discerning area relationships using cladograms, new inference methods are needed to bring more kinds of data to bear on questions about the geographic history of lineages. Here we describe a likelihood framework for inferring the evolution of geographic range on phylogenies that models lineage dispersal and local extinction in a set of discrete areas as stochastic events in continuous time. Unlike existing methods for estimating ancestral areas, such as dispersal-vicariance analysis, this approach incorporates information on the timing of both lineage divergences and the availability of connections between areas (dispersal routes). Monte Carlo methods are used to estimate branch-specific transition probabilities for geographic ranges, enabling the likelihood of the data (observed species distributions) to be evaluated for a given phylogeny and parameterized paleogeographic model. We demonstrate how the method can be used to address two biogeographic questions: What were the ancestral geographic ranges on a phylogenetic tree? How were those ancestral ranges affected by speciation and inherited by the daughter lineages at cladogenesis events? For illustration we use hypothetical examples and an analysis of a Northern Hemisphere plant clade (Cercis), comparing and contrasting inferences to those obtained from dispersal-vicariance analysis. Although the particular model we implement is somewhat simplistic, the framework itself is flexible and could readily be modified to incorporate additional sources of information and also be extended to address other aspects of historical biogeography.

Biological Evolution↗

Epidemiology of influenza C virus in man: multiple evolutionary lineages and low rate of change.

The nucleotide sequences of nonstructural protein (NS) genes of human influenza C viruses isolated between 1947 and 1983 were determined and compared. Assuming constant evolutionary rates, the extent of nucleotide differences among NS genes does not correspond to the isolation years of the strains. This suggests that more than one gene lineage is present in the population. Furthermore, examination of the eight C virus NS gene sequences by the maximum parsimony method (W. M. Fitch, 1971, Syst. Zool. 20, 406-416) yielded phylogenetic trees that were grossly different from those obtained using the hemagglutinin genes for the same eight isolates. This result is compatible with the idea of reassortment of genes in nature across lineages of influenza C viruses. The sequence analysis also suggests that nucleotide substitutions occur at a lower rate in the C virus NS genes than in influenza A virus NS genes.

Amino Acid Sequence↗

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum.

The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and interspecies relationships within Pisum.

Amino Acid Sequence↗

Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae.

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5'rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.

Base Sequence↗

Structural genomics sheds light on protein functions and remote homologs across the insect tree of life.

Protein structure bridges the sequence-function relationship, enabling deep exploration of biological processes across diverse organisms. Insects, the most diverse animal lineage, accounting for over 50% of all described animal species, provide an exceptional system for exploring sequence-structure-function relationships. Here, we reconstructed a comprehensive and well-resolved phylogeny of 4854 insects, spanning all orders. Leveraging this framework, we created an atlas of 13.29 million predicted protein structures from 824 representative species, including 11.63 million newly predicted structures. Structural clustering revealed that proteins with divergent sequences but similar structures could be effectively grouped together. Structural similarity searches against proteins with well-characterized functions yielded annotations for 7.61 million insect proteins, including up to 14% of previously unannotated proteins. We further identified 750 million remote homologs between insect proteins, many of which trace back to ancient branches of the insect phylogeny. Remarkably, despite extensive sequence divergence, cGAS-like receptors (cGLRs) were structurally conserved across all 824 insects. Experimental assays demonstrated that these structurally identified cGLRs play a crucial role in antiviral defense in the yellow fever mosquito. Our findings highlight the significance of structural genomics for understanding protein function and evolution across the tree of life.

Animals↗

Molecular phylogeography, intraspecific variation and the conservation of tree species.

Tree species are becoming the focus of increasing conservation concern, with some 9000 species now threatened globally. Studies of intraspecific variation can contribute to the development of conservation strategies, by identifying appropriate units for conservation. The recent application of molecular techniques to a variety of tree species has highlighted a far higher degree of population differentiation than indicated by previous isozyme analyses, a result consistent with theoretical predictions. Analysis of the geographic distribution of cpDNA lineages has also enabled current patterns of population differentiation to be related to postglacial migration routes from different forest refugia. Such results highlight the importance of refugial areas for conservation of intraspecific variation in tree species.

Journal Article↗

Analysis of puumala hantavirus genome in patients with nephropathia epidemica and rodent carriers from the sites of infection.

Reverse transcription-polymerase chain reaction (RT-PCR) followed by sequence and phylogenetic analyses were used to study specimens from nine Finnish nephropathia epidemica (NE) patients admitted to hospital during the epidemic in winter 1996-1997. Blood samples from six patients were found to be positive for the partial M- and/or S-segment sequences of Puumala hantavirus (PUUV). Analyses of these sequences (nt 2168-2610 for the M segment, and nt 819-1082 for the S segment) revealed six distinct PUUV strains showing highest similarity to previously described PUUV strains from Finland: 90-95% for the S segment, and 90-99% for the M segment. Accordingly, on the phylogenetic trees calculated for both viral segments, all six human strains were placed within the Finnish genetic lineage of PUUV. Attempts were made to trace five RT-PCR-positive patients to local bank voles (Clethrionomys glareolus) infected with wild-type PUUV, and for two patients a comparative analysis of human- and rodent-originated viral sequences was undertaken. Whereas in the first case the differences between the sequences were substantial (5. 7% for the S segment, and 10.8%, for the M segment), in the other case the M segment sequence recovered from the clinical specimen was 100% identical to three sequences recovered from rodent lungs, and the S sequences differed by one silent substitution only. This is the first finding of virtually identical PUUV sequences in an NE patient and a natural rodent host from the site of infection.

Adolescent↗

Unexpected conservation of the X-linked color vision gene in nocturnal prosimians: evidence from two bush babies.

Bush babies have had a long history of nocturnal life and it would be interesting to know whether their color vision genes have become degenerate. Therefore, we used PCR techniques to sequence the X-linked pigment gene of two of these nocturnal prosimians: Galago senegalensis and Otolemur garnettii. Southern hybridization of genomic DNA of G. senegalensis showed a single X-linked pigment gene. Interestingly, the deduced pigment sequences of the two bush babies are identical. By comparing the X-linked pigments of bush baby, human, squirrel monkey, and marmoset, 38 variable positions were identified. At those positions that may cause a spectral shift, the bush baby pigment has identical or biochemically similar residues to those of the marmoset cone pigment with a spectral peak of 543 nm. This result is consistent with the estimate of 544-545 nm for the spectral peak of the X-linked pigment of Otolemur crassicaudatus, which is closely related to Otolemur garnettii. The neighbor-joining tree of mammalian X-linked pigments showed a significantly shorter branch in the bush baby lineage than in other primate lineages. A relative rate test showed that the nonsynonymous substitution rate of the bush baby X-linked pigment gene is about three times slower than that of the human red pigment gene, though the synonymous substitution rates of the two genes are similar. The slower nonsynonymous rate in the bush baby lineage suggests that the bush baby X-linked pigment gene is under functional constraints, in spite of its nocturnal life. Two radical changes at positions in the intradiskal surface next to the sixth transmembrane domain were observed in the X-linked cone pigment of bush babies but not in other primates. They are changes from Ala to Ser and from Asn to His, which are similar in function to the corresponding residues in rhodopsins. These two changes may be of importance for dim light sensitivity, which is consistent with our proposal that the evolution of the bush baby X-linked pigment gene is under selective pressure. In addition, the 2.5% divergence in introns 2 and 5 of the X-linked pigment gene between the two bush babies supports their classification into two separate genera.

Adaptation, Physiological↗

Concordant evolution of a symbiont with its host insect species: molecular phylogeny of genus Glossina and its bacteriome-associated endosymbiont, Wigglesworthia glossinidia.

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the gamma subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species.

Animals↗

History of discovery of the first hyperthermophiles.

Hyperthermophiles, growing optimally at 80 degrees C and above had been discovered in 1981. They represent the upper temperature border of life and are found within high temperature environments. In their basically anaerobic surroundings, they gain energy mainly by inorganic redox reactions. Within the phylogenetic tree, hyperthermophiles occupy all the short deep branches closest to the root. The earliest archaeal phylogenetic lineage is represented by the extremely tiny members of the novel kingdom of Nanoarchaeota.

Anaerobiosis↗

The diversity of retroelements in diploid and allotetraploid Brassica species.

Using universal PCR primers, some 80 fragments of retroelement reverse transcriptase genes were isolated from 16 accessions of the three diploid and three derived allotetraploid species of Brassica in the triangle of U. Sequence analysis showed that the Ty1/copia and LINE-like elements were distinct, while a third clade could be sub-divided into Ty3/gypsy, Athila and virus-like branches, providing evidence that there are multiple sub-lineages within this group normally considered to be gypsy-like elements in plants. The parsimony trees showed no branches correlating with the known genome relationships for the six diploid and allotetraploid Brassica species, probably because members of the element families were present in the common ancestor of the Brassica and, unlike other repetitive sequences, there is no evidence for genome-wide homogenization, although convergent evolution or horizontal transfer cannot be ruled out. Southern hybridization suggested some sub-families were amplified in individual species. The data show that retroelement sequence data do not allow inference of phylogeny, but knowledge of evolution of such abundant sequences assists in exploitation and interpretation of data from other species including models with much smaller genomes and may provide markers.

Blotting, Southern↗

Molecular characterization of a partitivirus from Ophiostoma himal-ulmi.

The complete nucleotide sequences of two double-stranded (ds) RNA molecules, S1 (1,744 bp) and S2 (1,567 bp), isolated from an isolate HP62 of the Himalayan Dutch elm disease fungus, Ophiostoma himal-ulmi, were determined. RNA S1 had the potential to encode a protein, P1, of 539 amino acids (62.7 kDa), which contained sequence motifs characteristic of RNA-dependent RNA polymerases (RdRps). A database search showed that P1 was closely related to RdRps of members of the genus Partitivirus in the family Partitiviridae. RNA S2 had the potential to encode a protein, P2, of 430 amino acids (46.3 kDa), which was related to capsid proteins of members of the genus Partitivirus. Virus particles isolated from isolate HP62 were shown to be isometric with a diameter of 30 nm, and to contain dsRNAs S1 and S2 and a single capsid protein of 46 kDa. N-terminal sequencing of tryptic peptides derived from the capsid protein proved unequivocally that it is encoded by RNA S2 and corresponds to protein P2. It is concluded that O. himal-ulmi isolate HP62 contains a new member of the genus Partitivirus, which is designated Ophiostoma partitivirus 1. A phylogenetic tree of RdRps of members of the family Partitiviridae showed that there are least two RdRp lineages of viruses currently classified in the genus Partitivirus. One of these lineages contained viruses with fungal hosts and viruses with plant hosts, raising the possibility of horizontal transmission of partitiviruses between plants and fungi. The partitivirus RdRp and capsid proteins appear to have evolved in parallel with the capsid proteins evolving much faster than the RdRps.

Amino Acid Sequence↗