Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.     

Phylogenetics in Caenorhabditis elegans: an analysis of divergence and outcrossing

This study establishes a phylogenetic framework for the natural geographic isolates of the widely studied nematode species Caenorhabditis elegans. Virtually complete mitochondrial genomes are sequenced from 27 C. elegans natural isolates to characterize mitochondrial divergence patterns and to investigate the evolutionary history of the C. elegans hermaphrodite lineages. Phylogenetic analysis of mitochondrial sequences reveals the presence of two major C. elegans hermaphrodite clades (designated clade I and clade II). Fifty-six nuclear loci, widely distributed across the five autosomes and the X chromosome, are also analyzed in a subset of the C. elegans isolates to evaluate nuclear divergence patterns and the extent of mating between different strains. A comparison of the phylogenetic tree derived from mitochondrial data with the phylogenetic tree derived from nuclear data reveals only one inconsistency in the distribution of isolates into clades I and II, suggesting that mating between divergent C. elegans strains is an infrequent event in the wild.     

Nuclear ribosomal ITS sequences and phylogeny in East AsianAconitum subgenusAconitum (Ranunculaceae), with special reference to extensive polymorphism in individual plants

Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) were used to examine the phylogeny of East Asian aconites. Individual aconites were discovered to contain as many as eight different ITS sequences after cloning and PCR-SSCP (single-stranded conformational polymorphisms) analysis. We identified eight putative ITS pseudogenes from four taxa with low predicted secondary structure stability and high substitution rates. Maximum likelihood (ML) and neighbor-joining (NJ) methods were used for phylogenetic reconstruction. The ITS trees agree with the previous chloroplast DNA (cpDNA) tree for the vast majority of the taxa. We found two East Asian clades in the ITS trees: 1) a clade with the Chinese diploid,Aconitum volubile and East Asian tetraploids, and 2) a clade of East Asian diploids and Siberian tetraploids. In the former clade, most tetraploid taxa appear to be polyphyletic; sequences from individual plants did not correspond to recognized taxonomic units. This indicates a recent divergence of the East Asian tetraploids.     

Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.     

Molecular epidemiology of A/H3N2 and A/H1N1 influenza virus during a single epidemic season in the United States

To determine the spatial and temporal dynamics of influenza A virus during a single epidemic, we examined whole-genome sequences of 284 A/H1N1 and 69 A/H3N2 viruses collected across the continental United States during the 2006-2007 influenza season, representing the largest study of its kind undertaken to date. A phylogenetic analysis revealed that multiple clades of both A/H1N1 and A/H3N2 entered and co-circulated in the United States during this season, even in localities that are distant from major metropolitan areas, and with no clear pattern of spatial spread. In addition, co-circulating clades of the same subtype exchanged genome segments through reassortment, producing both a minor clade of A/H3N2 viruses that appears to have re-acquired sensitivity to the adamantane class of antiviral drugs, as well as a likely antigenically distinct A/H1N1 clade that became globally dominant following this season. Overall, the co-circulation of multiple viral clades during the 2006-2007 epidemic season revealed patterns of spatial spread that are far more complex than observed previously, and suggests a major role for both migration and reassortment in shaping the epidemiological dynamics of human influenza A virus.     

基于细胞核rDNA ITS片段的水青冈属的分子系统发育

对山毛榉科水青冈属6种、1亚种、1栽培变种的ITS区片段进行了测序和分析,并对其中2个具有ITS序列多态性的分类群进行了ITS区克隆。水青冈属ITS系统发育树聚成两支,位于基部的是分布于北美的大叶水青冈,另一分支则包括了欧洲和东亚的类群。在欧洲和东亚分支中,又包括两支,其中日本北部的波叶水青冈位于基部,台湾水青冈和欧亚大陆的水青冈形成另外一支。ITS区分析与现行的水青冈属基于形态学性状的属下分类系统有一定差异,而与本属现存物种的地理分布格局较为一致。各类群间TIS区序列差异较小,显示属内现存物种的分化时间不是太长。     

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8⁺ T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8⁺ T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8⁺ T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8⁺ T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.     

Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies.

The reactivities of a panel of 14 monoclonal antibodies (MAbs) with monomeric gp120 derived from 67 isolates of human immunodeficiency virus type 1 of clades A through F were assessed by using an antigen-capture enzyme-linked immunosorbent assay. The MAbs used were all raised against gp120 or gp120 peptides from clade B viruses and were directed at a range of epitopes relevant to human immunodeficiency virus type 1 neutralization: the V2 and V3 loops, discontinuous epitopes overlapping the CD4-binding site, and two other discontinuous epitopes. Four of the five V3 MAbs showed modest cross-reactivity within clade B but very limited reactivity with gp120s from other clades. These reactivity patterns are consistent with the known primary sequence requirements for the binding of these MAbs. One V3 human MAb (19b), however, was much more broadly reactive than the others, binding to 19 of 29 clade B and 10 of 12 clade E gp120s. The 19b epitope is confined to the flanks of the V3 loop, and these sequences are relatively conserved in clade B and E viruses. In contrast to the limited reactivity of V3 MAbs, CD4-binding site MAbs were much more broadly reactive across clades, two of these MAbs (205-46-9 and 21h) being virtually pan-reactive across clades A through F. Another human MAb (A-32) to a discontinuous epitope was also pan-reactive. The CD4-binding site is strongly conserved between clades; but when considering the epitopes near the CD4-binding site, clade D gp120 appears to be the most closely related to clade B and clade E appears to be the least related. A tentative rank order for these epitopes is B/D-A/C-E/F. V2 MAbs reacted sporadically within and between clades, and no clear pattern was observable. While results from binding assays do not predict neutralization serotypes, they suggest that there may be antigenic subtypes related, but not identical, to the genetic subtypes.     

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and beta-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade (Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira-Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.     

High-throughput sequencing of six bamboo chloroplast genomes: phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae)

Background

Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction) of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies.

Methodology/Principal Findings

Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp) genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with ≥0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae.

Conclusions/Significance

The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among three clades of temperate woody bamboos, even with complete cp genome sequences, suggests that these lineages may have diverged very rapidly.     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.     

Phylogenetic analysis based on full genome sequencing of Italian tomato spotted wilt virus isolates identified in “Roggianese” sweet pepper and chilli pepper

A study aimed at defining population structure of Italian tomato spotted wilt virus (TSWV) isolates was performed. Full genome sequencing of six TSWV isolates found in two Italian regions (two from Latium: Lazio 17 and Tarquinia; and four from Calabria: PepCal 10, 12, 22 and 24) were assembled. Identity percentages in nucleotide sequence among these TSWV isolates are here provided. The six full length genome sequences were compared with other two Italian isolates (p105 and p202/3WT) already fully sequenced, as well as full TSWV genomes that could be retrieved from GenBank. Phylogenetic analysis, performed in concatenated sequences and for each gene of each genome segment (L, M and S), confirmed the presence of two clades, namely A-like and D-like. In particular, the phylogenetic tree based on segment L grouped all the newly sequenced TSWV isolates in D-like clade. In the M segment phylogenetic tree, all our TSWV isolates shifted in the A-like clade. Isolates separation was not correlated to their geographical origin in phylogenetic study of distinct ORFs encoded by the RNA S segment. In fact, in nucleocapsid-encoding phylogenetic tree, PepCal 10 and 22 grouped in an A-like clade with p105, PepCal 12 and 24 in a D-like clade with p202/3WT, whereas Lazio 17 and Tarquinia in a third well distinct group. NSs tree displayed only PepCal 10 with p105 in A-like clade, whereas PepCal 12, 22, 24 with p202/3WT in D-like subclade; and isolates from Latium grouped a separated clade adjacent to D-like isolates. Additional analysis on putative reassortment events showed that TSWV Calabrian isolates likely originated from a reassortment event in M RNA and other in S RNA with p105 as major parent. Recombination events were detected in isolates from Latium in L and S RNAs with Chinese isolates as putative major parent.     

Phylogenetic analysis and molecular signatures defining a monophyletic clade of heterocystous cyanobacteria and identifying its closest relatives

Detailed phylogenetic and comparative genomic analyses are reported on 140 genome sequenced cyanobacteria with the main focus on the heterocyst-differentiating cyanobacteria. In a phylogenetic tree for cyanobacteria based upon concatenated sequences for 32 conserved proteins, the available cyanobacteria formed 8–9 strongly supported clades at the highest level, which may correspond to the higher taxonomic clades of this phylum. One of these clades contained all heterocystous cyanobacteria; within this clade, the members exhibiting either true (Nostocales) or false (Stigonematales) branching of filaments were intermixed indicating that the division of the heterocysts-forming cyanobacteria into these two groups is not supported by phylogenetic considerations. However, in both the protein tree as well as in the 16S rRNA gene tree, the akinete-forming heterocystous cyanobacteria formed a distinct clade. Within this clade, the members which differentiate into hormogonia or those which lack this ability were also separated into distinct groups. A novel molecular signature identified in this work that is uniquely shared by the akinete-forming heterocystous cyanobacteria provides further evidence that the members of this group are specifically related and they shared a common ancestor exclusive of the other cyanobacteria. Detailed comparative analyses on protein sequences from the genomes of heterocystous cyanobacteria reported here have also identified eight conserved signature indels (CSIs) in proteins involved in a broad range of functions, and three conserved signature proteins, that are either uniquely or mainly found in all heterocysts-forming cyanobacteria, but generally not found in other cyanobacteria. These molecular markers provide novel means for the identification of heterocystous cyanobacteria, and they provide evidence of their monophyletic origin. Additionally, this work has also identified seven CSIs in other proteins which in addition to the heterocystous cyanobacteria are uniquely shared by two smaller clades of cyanobacteria, which form the successive outgroups of the clade comprising of the heterocystous cyanobacteria in the protein trees. Based upon their close relationship to the heterocystous cyanobacteria, the members of these clades are indicated to be the closest relatives of the heterocysts-forming cyanobacteria.     

Molecular evolutionary dynamics of cytochrome b in strepsirrhine primates: the phylogenetic significance of third-position transversions

DNA sequences of the complete cytochrome b gene are shown to contain robust phylogenetic signal for the strepsirrhine primates (i.e., lemurs and lorises). The phylogeny derived from these data conforms to other molecular studies of strepsirrhine relationships despite the fact that uncorrected nucleotide distances are high for nearly all intrastrepsirrhine comparisons, with most in the 15%-20% range. Cytochrome b sequences support the hypothesis that Malagasy lemuriforms and Afro-Asian lorisiforms each comprise clades that share a sister- group relationship. A study (Adkins and Honeycutt 1994) of the cytochrome c oxidase subunit II (COII) gene placed one Malagasy primate (Daubentonia) at the base of the strepsirrhine clade, thereby suggesting a diphyletic Lemuriformes. The reanalysis of COII third- position transversions, either alone or in combination with cytochrome b third-position transversions, however, yields a tree that is congruent with phylogenetic hypotheses derived from cytochrome b and other genetic data sets.      

Epidemics and Frequent Recombination within Species in Outbreaks of Human Enterovirus B-Associated Hand,Foot and Mouth Disease in Shandong China in 2010 and 2011

The epidemiology and molecular characteristics of human enterovirus B (HEV-B) associated with hand, foot and mouth disease (HFMD) outbreaks in China are not well known. In the present study, we tested 201 HEV isolates from 233 clinical specimens from patients with severe HFMD during 2010–2011 in Linyi, Shandong, China. Of the 201 isolates, 189 were fully typed and 18 corresponded to HEV-B species (six serotypes CVA9, CVB1, CVB4, Echo 6, Echo 25 and Echo 30) using sensitive semi-nested polymerase chain reaction analysis of VP1 gene sequences. Phylogenetic analysis based on the VP1 region showed that eight E30SD belonged to a novel sub-genogroup D2; E25SD belonged to a novel sub-genogroup D6; E6SD belonged to sub-lineage C6 and five CVB1SD belonged to subgroup 4C; and B4SD belonged sub-lineage D2. The full viral genomes of the CVB1SD, E6SD, E25SD and E30SD isolates were sequenced. Analysis of phylogenetic and similarity plots indicated that E25SD recombined with E25-HN-2, E30FDJS03 and E4AUS250 at noncontiguous P2A–P3D regions, while E30SD, E30FDJ03, E25-HN-2 and E9 DM had shared sequences in discrete regions of P2 and P3. Both E6SD and B1SD shared sequences with E1-HN, B4/GX/10, B5-HN, and A9-Alberta in contiguous regions of most of P2 and P3. Genetic algorithm recombination detection analysis further confirmed the existence of multiple potential recombination points. In conclusion, analysis of the complete genomes of E25SD, E30SD, CVB1SD and E6SD isolated from HFMD patients revealed that they formed novel subgenogroup. Given the prevalence and recombination of these viruses in outbreaks of HFMD, persistent surveillance of HFMD-associated HEV-B pathogens is required to predict potential emerging viruses and related disease outbreaks.     

Supermatrix data highlight the phylogenetic relationships of photosynthetic stramenopiles

Molecular data had consistently recovered monophyletic classes for the heterokont algae, however, the relationships among the classes had remained only partially resolved. Furthermore, earlier studies did not include representatives from all taxonomic classes. We used a five-gene (nuclear encoded SSU rRNA; plastid encoded rbcL, psaA, psbA, psbC) analysis with a subset of 89 taxa representing all 16 heterokont classes to infer a phylogenetic tree. There were three major clades. The Aurearenophyceae, Chrysomerophyceae, Phaeophyceae, Phaeothamniophyceae, Raphidophyceae, Schizocladiophyceae and Xanthophyceae formed the SI clade. The Chrysophyceae, Eustigmatophyceae, Pinguiophyceae, Synchromophyceae and Synurophyceae formed the SII clade. The Bacillariophyceae, Bolidophyceae, Dictyochophyceae and Pelagophyceae formed the SIII clade. These three clades were also found in a ten-gene analysis. The approximately unbiased test rejected alternative hypotheses that forced each class into either of the other two clades. Morphological and biochemical data were not available for all 89 taxa, however, existing data were consistent with the molecular phylogenetic tree, especially for the SIII clade.     

Geographic variation and diversity of the cytochrome b gene in wild populations of medaka (Oryzias latipes) from Korea and China

We analyzed the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b (cyt b) gene in wild populations of medaka from Korea and China. We surveyed 258 wild specimens from 75 different sites, and identified 17 mitotypes. Sequencing analysis of the complete cyt b gene (1141-bp) was subsequently carried out to infer the phylogenetic relationships among these mitotypes. Phylogenetic trees indicated two major clades, D and E, which were different from the Japanese clades (A, B and C). These two clades were completely identical to two clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of the mitotypes in clades D and E was consistent with the China-West Korean Population and the East Korean Population as defined by allozymic and karyological analyses. This agreement among different analyses suggests long-term isolation between the two groups. In the region where the distributions of two major clades overlapped, a limited extent of gene flow was observed. These results suggested the existence of some reproductive isolation mechanisms between the two clades, or the introgression between them followed by a random drift in each local population. Furthermore, clade D was subdivided into three subclades (D-I to D-III). The phylogenetic relationship and distribution pattern of subclade D-II suggested a dispersal event of medaka from China to southwest Korea. Our results also showed that the East Korean Population has recently expanded its distribution area because little diversity was observed in clade E.     

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (相似文献   

Biogeography and evolution of flower color in Veratrum (Melanthiaceae) through inference of a phylogeny based on multiple DNA markers

Veratrum (Melanthiaceae) comprises ca. 27 species with highly variable morphology. This study aims to construct the molecular phylogeny of this genus to infer its floral evolution and historical biogeography, which have not been examined in detail before. Maximum parsimony, maximum likelihood, and Bayesian analyses were performed on the separate and combined ITS, trnL-F, and atpB-rbcL sequences to reconstruct the phylogenetic tree of the genus. All Veratrum taxa formed a monophyletic group, within which two distinct clades were distinguished: species with white-to-green perianth formed one highly supported clade, and the species with black-purple perianth constituted another highly supported clade. Phylogenetic inference on flower color evolution suggested that white-to-green perianth was a plesiomorphic state and black-purple perianth was apomorphic for Veratrum. When species distribution areas were traced as a multi-state character, parsimonious optimization inferred that Veratrum possibly originated in East Asia. Our study confirmed previous phylogenetic and taxonomic suggestions on this genus and provided a typical example of plant radiation across the Northern Hemisphere.     

Genetic diversity of Usutu virus

Usutu virus is a mosquito-borne virus first isolated from Culex naevei in South Africa in 1959. The first emergence of Usutu virus outside Africa was recorded in Austria. Here, a phylogenetic analysis targeting the E5 and NS5 genes was carried out on the viral strains circulating in Europe. The NS5 gene tree showed two main clades, one of which included the Italian sequences. In the E gene tree all sequences grouped into the same main clade, with sequences from Austria divided into two separate clusters. Only sites under negative selective pressure were found in E and NS5 proteins. The results suggest that Usutu virus circulating in Europe has a degree of genetic diversity higher than expected and that infection may arise from different sources.