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Origin and evolution of the slime molds (Mycetozoa)

The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid), and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. Ribosomal RNA trees show the myxogastrid and dictyostelid slime molds as unrelated early branching lineages, but actin and beta-tubulin trees place them together as a single coherent (monophyletic) group, closely related to the animal-fungal clade. We have sequenced the elongation factor-1alpha genes from one member of each division of the Mycetozoa, including Dictyostelium discoideum, for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, with the myxogastrid and dictyostelid slime molds most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals + fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four that place these taxa within the "crown" of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and we emphasize the importance of developing multiple sequence data sets.

Amino Acid Sequence↗

Molecular evolution of Vibrio pathogenicity island-2 (VPI-2): mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates.

Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the aetiological agent of cholera, a disease that is endemic in much of Southern Asia. The 57.3 kb Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V. cholerae O1 and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism gene cluster (VC1773-VC1783), a neuraminidase (VC1784) and a gene cluster that shows homology to Mu phage. In this study, a 14.1 kb region of VPI-2 comprising ORFs VC1773 to VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates examined. The VPI-2 region in V. mimicus was inserted adjacent to a serine tRNA similar to VPI-2 in V. cholerae. In 11 of the 17 V. mimicus isolates examined, an additional 5.3 kb region encoding VC1758 and VC1804 to VC1809 was present adjacent to VC1787. The evolutionary history of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh), among V. cholerae and V. mimicus isolates. Both gene trees showed an overall congruence; on both gene trees V. cholerae O1 and O139 serogroup isolates clustered together, whereas non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering of strains within the branches. One exception was noted: on the mdh gene tree, V. mimicus sequences formed a distinct divergent lineage from V. cholerae sequences; however, on the nanH gene tree, V. mimicus clustered with V. cholerae non-O1/non-O139 isolates, suggesting horizontal transfer of this region between these species.

Bacterial Proteins↗

Analysis of differentiation of mouse hemopoietic stem cells in culture by sequential replating of paired progenitors.

Blast cell colonies seen in cultures of spleen cells from 5-fluorouracil-treated mice provide a highly enriched population of primitive hemopoietic progenitors. Our recent studies of the differentiation potentials of the paired daughter cells of these progenitors showed different patterns of differentiation in the colonies produced by the separated daughter cells. In this study, we carried out sequential micromanipulation of paired progenitors followed by cytologic examinations of the colonies derived from these progenitors. Of the total 94 evaluable cultures, consisting of three or more colonies, 52 consisted of macrophage colonies and one consisted of megakaryocyte colonies. In the remaining 41 cultures, diverse combinations of colonies revealing heterogeneous compositions of cell lineages were identified. Presumptive genealogic trees of the differentiation of hemopoietic progenitors constructed for the latter group of cultures suggested that monopotent progenitors may be derived from pluripotent progenitors in two ways: (1) directly during one cell division of pluripotent cells or (2) as a result of progressive lineage restriction during successive division of the pluripotent progenitors. The results also suggested that some of the oligopotent progenitors are capable of limited self-renewal.

Animals↗

Molecular phylogeny of the alphaherpesvirinae subfamily and a proposed evolutionary timescale.

Phylogenetic trees were derived for the Alphaherpesvirinae subfamily of the Herpesviridae using molecular sequences. Sequences from the families of genes encoding glycoprotein B, thymidine kinase, S region protein kinase, immediate-early transcriptional regulator IE175 and ribonucleotide reductase large subunit were examined by means of both maximum parsimony and distance methods, and for both protein and DNA alignments. Trees obtained were evaluated by bootstrap analysis. A clear consensus tree was obtained, with most detail coming from 14 sequences in the glycoprotein B gene set. The tree showed two avian viruses branching first from the lineage leading to the mammalian alphaherpesviruses. The mammalian viruses were split into two groups, which corresponded to the Simplexvirus and Varicellovirus genera. A timescale for events in alphaherpesvirus evolution was tested, based on the proposition that most of the lineages arose by ancient cospeciation with hosts. The virus phylogenetic tree was unambiguously compatible with cospeciation for ten of the 12 mammalian viruses. The tree was also supported by demonstration of an approximate proportionality between magnitudes of pairwise divergences of viral sequences and times since lineages of corresponding pairs of hosts split. On the basis of this timescale it was estimated that the two mammalian alphaherpesvirus groups diverged around the period of the mammalian radiation, and that alphaherpesviral genome sequences have evolved faster than those of mammals by a factor of one to two orders of magnitude.

Alphaherpesvirinae↗

Mitochondrial DNA evolution in primates: transition rate has been extremely low in the lemur.

Based on mitochondrial DNA (mt-DNA) sequence data from a wide range of primate species, branching order in the evolution of primates was inferred by the maximum likelihood method of Felsenstein without assuming rate constancy among lineages. Bootstrap probabilities for the maximum likelihood tree topology among alternatives were estimated without performing a maximum likelihood estimation for each resampled data set. Variation in the evolutionary rate among lineages was examined for the maximum likelihood tree by a method developed by Kishino and Hasegawa. From these analyses it appears that the transition rate of mtDNA evolution in the lemur has been extremely low, only about 1/10 that in other primate lines, whereas the transversion rate does not differ significantly from that of other primates. Furthermore, the transition rate in catarrhines, except the gibbon, is higher than those in the tarsier and in platyrrhines, and the transition rate in the gibbon is lower than those in other catarrhines. Branching dates in primate evolution were estimated by a molecular clock analysis of mtDNA, taking into account the rate of variation among different lines, and the results were compared with those estimated from nuclear DNA. Under the most likely model, where the evolutionary rate of mtDNA has been uniform within a great apes/human clade, human/chimpanzee clustering is preferred to the alternative branching orders among human, chimpanzee, and gorilla.

Animals↗

An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.

Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution.

Algorithms↗

DNA-DNA hybridization-based phylogeny for "higher" nonpasserines: reevaluating a key portion of the avian family tree.

A matrix of delta T mode values for 10 birds, including 9 nonpasserines and a suboscine passerine flycatcher, was generated by DNA-DNA hybridization. Within the most derived lineages, all bootstrapped and jackknifed FITCH trees lend strong support to sister-groupings of the two swift families, of hummingbirds to swifts, and of these to a clade containing both owls and night-hawks. The outgroup duck roots the tree between the woodpecker (Piciformes) and the remaining taxa, indicating that Piciformes are among the earliest branches within nonpasserines. However, the succeeding branches to kingfisher, mousebird, and suboscine passerine flycatcher are based on short internodes that are poorly supported by bootstrapping and that give inconsistent results in jackknifing. Although these 3 orders may have arisen through rapid or near-simultaneous divergence, placement of the "advanced" Passeriformes deep within a more "primitive" radiation indicates that nonpasserines are paraphyletic, echoing the same distinction for reptiles with respect to their advanced descendants. Despite significant rate variation among different taxa, these results largely concur with those obtained with the same technique by Sibley and Ahlquist, who used the delta T50H measure and UPGMA analysis. This agreement lends credence to some of their more controversial claims.

Animals↗

Molecular data suggest that microsporidian parasites in freshwater snails are diverse.

Microsporidian parasites infect almost all invertebrate and vertebrate hosts and have significant effects on individual and population fitness. Phylogenetic analysis demonstrates that the phylum is highly divergent and that some lineages show strong associations with host taxa. We here examine the diversity and distribution of parasites in gastropod molluscs to test for host-parasite co-association. 16 populations representing 10 species of freshwater snails were screened using microsporidian specific small subunit rDNA primers. Four novel microsporidian parasite sequences were detected within populations of three host species from the genera Bulinus, Biomphalaria and Planorbis. Prevalence ranged from 5 to 84%. Phylogenetic analysis of these novel sequences reveals that they group together as a paraphyletic assemblage in the microsporidian tree basal to the two lineages containing the genera Encephalitozoon and Nosema. Preliminary observation of one microsporidian infection, show parasites distributed in all tissue systems of Bulinus globosus. However, infection is most prevalent in the digestive gland while also in the egg sacs, suggesting that the microsporidium is using a mixed strategy of horizontal and vertical transmission in this population.

Animals↗

Perspectives on future applications of experimental biology to evolution.

The first three decades of the subdiscipline of biology known as "molecular evolution" have generated large amounts of new information that illuminate the nature of evolutionary pattern and process. Major progress has been made in identifying primary sequence variation in genes and their protein products, initially from biochemically tractable systems (from large or culturable organisms and from highly-reiterated genes or highly-expressed gene products). In the 1980s, these techniques that had been limited to specialists, to relatively few representatives of the diversity of life, and to a small number of those organisms' genes, were extended through advances in molecular genetics and biochemistry, resulting in an explosion of molecular information and a proliferation of molecular trees. Studies of variation in molecular characters also were rarely linked with studies of anatomical, behavioral or ecological diversity. More sophisticated molecular genetic and biochemical techniques, currently being applied to long-standing questions in cell and developmental biology in model systems, should be applicable to more diverse lineages in the next decade. Molecular trees produced from one or more "housekeeping genes" can identify key lineages (species, populations, genomes or gene families) which, by comparison to model systems, may illuminate important aspects of higher level variability. Thus, the next phase of research in the field of molecular evolution should see greater linkage between studies of simple molecular and more complex developmental characters, and increased functional testing of genes and gene products in an evolutionary context. This review highlights some comparative experimental approaches that I believe will be most effective in extending our understanding of molecular evolution and better linking the field to other areas of science in the next few years.

Animals↗

Diversity estimates of microeukaryotes below the chemocline of the anoxic Mariager Fjord, Denmark.

Microbial communities of extreme environments have often been assumed to have low species richness. We analysed 18S rRNA gene signatures in a sample collected below the chemocline of the anoxic Mariager Fjord in Denmark, and from these data we computed novel parametric and standard nonparametric estimates of protistan phylotype richness. Our results indicate unexpectedly high richness in this environment: at the 99.5% phylotype definition, our most conservative estimate was 568 phylotypes (+/-114, standard error). Phylogenetic analyses revealed that the sequences collected cover the majority of described lineages in the eukaryotic domain. Out of 384 sequences analysed, 307 were identified as protistan targets, none of which was identical to known sequences. However, based on what is known about species that are phylogenetically related to the Mariager sequences, most of the latter seem to belong to strictly or facultative anaerobe organisms. We also found signatures that together with other environmental 18S rRNA gene sequences represent environmental clades of possibly high taxonomic levels (class to kingdom level). One of these clades, consisting exclusively of sequences from anoxic sampling sites, branches at the base of the eukaryotic evolutionary tree among the earliest eukaryotic lineages. Assuming eukaryotic evolution under oxygen-depleted conditions, these sequences may represent immediate descendants of early eukaryotic ancestors.

Colony Count, Microbial↗

Phylogenetics and evolution of the Daphnia longispina group (Crustacea) based on 12S rDNA sequence and allozyme variation.

Although members of the crustacean genus Daphnia have been the target of much research, there is little understanding of the group's evolutionary history. We addressed this gap by inferring a phylogeny for one of the major species groups (longispina) using nucleotide sequence variation of a 525-bp segment of the mitochondrial 12S rDNA and allozyme variation at 21 loci. We identified the major lineages and their relationships, assessed the phylogenetic utility of the few morphological characters in the group, and examined Daphnia phylogeography. Nuclear and mtDNA phylogenies were generally concordant in recognizing the same four species complexes. An exception was the position of Daphnia galeata mendotae. The allozyme tree paired this species with the Daphnia rosea lineage, whereas the mtDNA trees grouped D. g. mendotae with Daphnia galeata galeata. This discordance was consistent with the reticulate evolution of nuclear genes supporting the hypothesis that D. g. mendotae represents a case of homoploid hybrid speciation. Striking morphological stasis in the longispina group was evidenced by its very limited morphological divergence over an estimated 100 MY, and by the unusual transitional saturation of the conservative 12S rRNA gene within a species group. Phylogenetic inference also provided evidence that similarities in cephalic crest shape likely resulted from convergent or parallel evolution among species. Endemism at the continental level was indicated for previously cosmopolitan species, but the estimated times of these divisions were inconsistent with vicariance events suggesting recent dispersal among continents. A significant role for divergent selection in new habitats during speciation was suggested by the neighboringly sympatric distributions of four sister species pairs over broad geographic areas.

Alleles↗

Molecular phylogeny, evolutionary rates, and divergence timing of the symbiotic dinoflagellate genus Symbiodinium.

Symbiotic dinoflagellates belonging to the genus Symbiodinium are found in association with a wide variety of shallow-water invertebrates and protists dwelling in tropical and subtropical coral-reef ecosystems. Molecular phylogeny of Symbiodinium, initially inferred using nuclear ribosomal genes, was recently confirmed by studies of chloroplastic and mitochondrial genes, but with limited taxon sampling and low resolution. Here, we present the first complete view of Symbiodinium phylogeny based on concatenated partial sequences of chloroplast 23S-rDNA (cp23S) and nuclear 28S-rDNA (nr28S) genes, including all known Symbiodinium lineages. Our data produced a well resolved phylogenetic tree and provide a strong statistical support for the eight distinctive clades (A-H) that form the major taxa of Symbiodinium. The relative-rate tests did not show particularly high differences between lineages and both analysed markers. However, maximum likelihood ratio tests rejected a global molecular clock. Therefore, we applied a relaxed molecular clock method to infer the divergence times of all extant lineages of Symbiodinium, calibrating its phylogenetic tree with the fossil record of soritid foraminifera. Our analysis suggests that Symbiodinium originated in early Eocene, and that the majority of extant lineages diversified since mid-Miocene, about 15 million years ago.

Animals↗

Perils of paralogy: using HSP70 genes for inferring organismal phylogenies.

Conserved genes have found their way into the mainstream of molecular systematics. Many of these genes are members of multigene families. A difficulty with using single genes of multigene families for phylogenetic inference is that genes from one species may be paralogous to those from another taxon. We focus attention on this problem using heat shock 70 (HSP70) genes. Using polymerase chain reaction techniques with genomic DNA, we isolated and sequenced 123 distinct sequences from 12 species of sharks. Phylogenetic analysis indicated that the sequences cluster with constituitively expressed cytoplasmic heat shock-like genes. Three highly divergent gene clades were sampled. A number of similar sequences were sampled from each species within each distinct gene clade. Comparison of published species trees with an HSP70 gene tree inferred using Bayesian phylogenetic analysis revealed several cases of gene duplication and differential sorting of gene lineages within this group of sharks. Gene tree parsimony based on the objective criteria of duplication and losses showed that previously published hypotheses of species relationships and two novel hypothesis based on Bayesian phylogenetics were concordant with the history of HSP70 gene duplication and loss. By contrast, two published hypotheses based on morphological data were not significantly different from the null hypothesis of a random association between species relatedness and the HSP70 gene tree. These results suggest that gene tree parsimony using data from multigene families can be used for inferring species relationships or testing published alternative hypotheses. More importantly, the results suggest that systematic studies relying on phylogenetic inferences from HSP70 genes may by plagued by unrecognized paralogy of sampled genes. Our results underscore the distinction between gene and species trees and highlight an underappreciated source of discordance between gene trees and organismal phylogeny, i.e., unrecognized paralogy of sampled genes.

Animals↗

Differentiation within the genus Leptocarabus (excl. L. kurilensis) in the Japanese Islands as deduced from mitochondrial ND5 gene sequences (Coleoptera, Carabidae).

The phylogenetic trees have been constructed for the mitochondrial ND5 gene sequences from the Japanese Leptocarabus ground beetles, which contain 101 specimens collected from nearly the complete distribution ranges of them consisting of five morphological species, i.e., Leptocarabus procerulus, L. kumagaii, L. hiurai, L. kyushuensis and L. arboreus. On the trees, there are recognized two major lineages, each of which is further divided into two or more sublineages. The phylogenetic lines are geographically linked. Two or more species occur in a single lineage, and the same species appear in different lines. We suggest that transformation from one type of morphology to another took place in parallel in various periods of evolution of the Japanese Leptocarabus. From the phylogenetic tree and the dating from the nucleotide substitution rate and the geohistorical data it is inferred that the ancestry of all the Japanese Leptocarabus species was derived from a protoform of L. kyushuensis inhabited the ancient Japan area, followed by separation into two lineages after split of the Japanese Islands from the Eurasian Continent. They then propagated distribution to occupy their own habitat ranges, during which the morphological transformation took place in some lineages.

Animals↗

A molecular perspective on the systematics and evolution of the genus Arvicanthis (Rodentia, Muridae): inferences from complete cytochrome b gene sequences.

Systematics of the genus Arvicanthis, the African unstriped grass rat, are somewhat controversial. Most recent taxonomic revisions list five to six species but the definition of some of these (Arvicanthis dembeensis, Arvicanthis nairobae, and Arvicanthis niloticus) is uncertain. The complete mitochondrial cytochrome b gene (1140 bp) was sequenced for 20 specimens from throughout the range of the genus to determine the intrageneric genetic structure, construct a molecular phylogeny, and evaluate classical taxonomies. Neighbor-joining and maximum parsimony analyses yielded identical phylogenetic trees that identify two major lineages: the first one (1) is composed of specimens usually referred to A. niloticus but representing several distinct species, and the other (2) is a complex including "true" A. niloticus from Egypt and northern West Africa as well as Arvicanthis abyssinicus, Arvicanthis dembeensis, and Arvicanthis somalicus. An analysis on a 357-bp fragment of the cytochrome b including published data on A. nairobae indicates that this taxon is part of clade (1). Calibration of the number of 3rd position transversion changes with the murid fossil record suggests that clades (1) and (2) diverged approximately 5 Myr ago. Arvicanthis niloticus as currently recognized is a paraphyletic association and this name should be restricted to the Egyptian and northern West African sample. We also suggest referring to A. dembeensis as A. niloticus, as our cytochrome b data do not support its recognition as a distinct species. Clade (1) is subdivided in three lineages, geographically corresponding to southern West, Central, and East Africa. The high genetic divergence detected between the Central African lineage and the other two lineages suggests that they probably represent separate species. Clade (2) experienced rapid cladogenetic events during the late Pliocene, with the A. somalicus lineage being the first to emerge, followed by the ancestor of A. abyssinicus and A. blicki. This period was characterized by significant climatic and environmental changes, such as the extension of open habitats, which might have provided a stimulus for speciation in this savanna-dwelling genus. Confrontation of our molecular results with chromosomal data shows a high degree of congruence between the two datasets.

Animals↗

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products.

The nature of the bcl-2 family of proto-oncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man--genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered.

Amino Acid Sequence↗

Phylogenetic calibration of the 5' terminal domain of large rRNA achieved by determining twenty eucaryotic sequences.

Due to their high information content and their particular mode of variation, large rRNA molecules potentially represent powerful indicators of phylogenetic relationships. Even partial sequences may suffice to generate reliable estimations, provided they correspond to well-chosen portions of the molecule. We have systematically analyzed a specific portion of the large rRNA (the region extending over nearly 400 nucleotides from the 5' end) as a general index of eucaryotic phylogeny. By means of fast and direct rRNA sequencing, we have determined the sequence of this region for 20 additional eucaryotes, including several representatives of each vertebrate class, an invertebrate metazoan (mussel), a fungus (Schizosaccharomyces pombe), and three higher plants. Comparative treatment of these new data and previously reported rRNA sequences shows that this region can serve as an indicator of eucaryotic phylogeny for evaluating both long-range and short-range relationships. Its conservative domains appear to possess a rather uniform rate of nucleotide changes in all the eucaryotic lineages analyzed and the phylogenetic tree we derived agrees with classical views.

Animals↗

Tick-borne encephalitis virus in Lithuania.

Tick-borne encephalitis (TBE) is a severe problem in Lithuania, indicated by the 171 to 645 serologically confirmed cases that occurred each year between 1993 and 1999. In the present report, the first isolation and partial genetic analysis of a Lithuanian TBE virus (TBEV) strain isolated from a patient's serum sample is described. The patient was bitten by a tick while visiting the Lazdijai district (Veisiejai forest) in the southernmost part of Lithuania, a geographical area where Ixodes ricinus but not Ixodes persulcatus ticks are known to be present. The E protein-encoding viral gene sequence (nt 74-1273) recovered from the TBEV isolate showed the closest similarity to previously characterized European strains of the Western TBEV subtype, including the prototype TBEV strain Neudoerfl and those from neighbouring Latvia. Accordingly, the Lithuanian isolate was placed within the Western genetic lineage of TBEV in phylogenetic trees.

Adult↗