Using PhyloSuite for molecular phylogeny and tree-based analyses.

Phylogenetic analysis has entered the genomics (multilocus) era. For less experienced researchers, conquering the large number of software programs required for a multilocus-based phylogenetic reconstruction can be somewhat daunting and time-consuming. PhyloSuite, a software with a user-friendly GUI, was designed to make this process more accessible by integrating multiple software programs needed for multilocus and single-gene phylogenies and further streamlining the whole process. In this protocol, we aim to explain how to conduct each step of the phylogenetic pipeline and tree-based analyses in PhyloSuite. We also present a new version of PhyloSuite (v1.2.3), wherein we fixed some bugs, made some optimizations, and introduced some new functions, including a number of tree-based analyses, such as signal-to-noise calculation, saturation analysis, spurious species identification, and etc. The step-by-step protocol includes background information (i.e., what the step does), reasons (i.e., why do the step), and operations (i.e., how to do it). This protocol will help researchers quick-start their way through the multilocus phylogenetic analysis, especially those interested in conducting organelle-based analyses.

Bayesian Phylogeographic Inference Suggests Japan as the Center for the Origin and Dissemination of Rice Stripe Virus.

Rice stripe virus (RSV) is one of the most important viral pathogens of rice in East Asia. The origin and dispersal of RSV remain poorly understood, but an emerging hypothesis suggests that: (i) RSV originates from Yunnan, a southwest province of China; and (ii) some places of eastern China have acted as a center for the international dissemination of RSV. This hypothesis, however, has never been tested rigorously. Using a data set comprising more than 200 time-stamped coat protein gene sequences of RSV from Japan, China and South Korea, we reconstructed the phylogeographic history of RSV with Bayesian phylogeographic inference. Unexpectedly, the results did not support the abovementioned hypothesis. Instead, they suggested that RSV originates from Japan and Japan has been the major center for the dissemination of RSV in the past decades. Based on these data and the temporal dynamics of RSV reported recently by another group, we proposed a new hypothesis to explain the origin and dispersal of RSV. This new hypothesis may be valuable for further studies aiming to clarify the epidemiology of RSV. It may also be useful in designing management strategies against this devastating virus.

Correction: Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

[This corrects the article DOI: 10.1371/journal.pone.0231961.].

ggmsa: a visual exploration tool for multiple sequence alignment and associated data.

The identification of the conserved and variable regions in the multiple sequence alignment (MSA) is critical to accelerating the process of understanding the function of genes. MSA visualizations allow us to transform sequence features into understandable visual representations. As the sequence-structure-function relationship gains increasing attention in molecular biology studies, the simple display of nucleotide or protein sequence alignment is not satisfied. A more scalable visualization is required to broaden the scope of sequence investigation. Here we present ggmsa, an R package for mining comprehensive sequence features and integrating the associated data of MSA by a variety of display methods. To uncover sequence conservation patterns, variations and recombination at the site level, sequence bundles, sequence logos, stacked sequence alignment and comparative plots are implemented. ggmsa supports integrating the correlation of MSA sequences and their phenotypes, as well as other traits such as ancestral sequences, molecular structures, molecular functions and expression levels. We also design a new visualization method for genome alignments in multiple alignment format to explore the pattern of within and between species variation. Combining these visual representations with prime knowledge, ggmsa assists researchers in discovering MSA and making decisions. The ggmsa package is open-source software released under the Artistic-2.0 license, and it is freely available on Bioconductor (https://bioconductor.org/packages/ggmsa) and Github (https://github.com/YuLab-SMU/ggmsa).

Genomic Epidemiology and Evolution of Scallion Mosaic Potyvirus From Asymptomatic Wild Japanese Garlic.

Scallion mosaic virus (ScaMV) belongs to the turnip mosaic virus phylogenetic group of potyvirus and is known to infect domestic scallion plants (Allium chinense) in China and wild Japanese garlic (Allium macrostemon Bunge) in Japan. Wild Japanese garlic plants showing asymptomatic leaves were collected from different sites in Japan during 2012-2015. We found that 73 wild Japanese garlic plants out of 277 collected plants were infected with ScaMV, identified by partial genomic nucleotide sequences of the amplified RT-PCR products using potyvirus-specific primer pairs. Sixty-three ScaMV isolates were then chosen, and those full genomic sequences were determined. We carried out evolutionary analyses of the complete polyprotein-coding sequences and four non-recombinogenic regions of partial genomic sequences. We found that 80% of ScaMV samples have recombination-like genome structure and identified 12 recombination-type patterns in the genomes of the Japanese ScaMV isolates. Furthermore, we found two non-recombinant-type patterns in the Japanese population. Because the wild plants and weeds may often serve as reservoirs of viruses, it is important to study providing the exploratory investigation before emergence in the domestic plants. This is possibly the first epidemiological and evolutionary study of a virus from asymptomatic wild plants.

Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

The phylogeographic history of tomato mosaic virus in Eurasia.

Tomato mosaic virus (ToMV) is a tobamovirus affecting solanaceous crops worldwide. The process of its emergence, however, is poorly understood. Here, Bayesian phylogenetic framework was employed to reconstruct the phylogeography of ToMV in Eurasia. The results showed that the ToMV in Europe, Middle East and East Asia has been evolving at a rate of 4.05 × 10-4 substitutions/site/year (95% credibility interval 2.43 × 10-4 - 5.62 × 10-4). Their most recent common ancestor (MRCA), most probably first appeared in Europe, was dated to around 1757 Common Era. The first introduction of ToMV into Middle East occurred in 1920s, with Europe as the source, while the first introduction of ToMV into East Asia occurred shortly afterwards, with Middle East as the source. From about 1950 onwards, inter-regional migrations of ToMV between Europe, Middle East and East Asia have been common. Overall, these data provide a glimpse into the phylogeographic history of ToMV in Eurasia.

The evolutionary history and global spatio-temporal dynamics of potato virus Y.

Potato virus Y (PVY) is a destructive plant pathogen that causes considerable losses to global potato and tobacco production. Although the molecular structure of PVY is well characterized, the evolutionary and global transmission dynamics of this virus remain poorly understood. We investigated the phylodynamics of the virus by analysing 253 nucleotide sequences of the genes encoding the third protein (P3), cylindrical inclusion protein (CI), and the nuclear inclusion protein (NIb). Our Bayesian phylogenetic analyses showed that the mean substitution rates of different regions of the genome ranged from 8.50 × 10-5 to 1.34 × 10-4 substitutions/site/year, whereas the time to the most recent common ancestor of PVY varied with the length of the genomic regions and with the number of viral isolates being analysed. Our phylogeographic analysis showed that the PVY population originated in South America and was introduced into Europe in the 19th century, from where it spread around the globe. The migration pathways of PVY correlate well with the trade routes of potato tubers, suggesting that the global spread of PVY is associated with human activities.

The Gene Flow Direction of Geographically Distinct Phytophthora infestans Populations in China Corresponds With the Route of Seed Potato Exchange.

Phytophthora infestans is a widespread destructive plant pathogen that causes economic losses worldwide to potato production. In this study, we sequenced four mitochondrial DNA gene sequences of 101 P. infestans isolates from five potato-growing regions in China to investigate the population structure and dispersal pattern of this pathogen. The concatenated mtDNA sequences in the populations showed high haplotype diversity, but low nucleotide diversity. Although there was a degree of spatial structure, our phylogeographic analyses support frequent gene flow between populations and the direction of gene flow, primarily from north to south, corresponds to the route of seed potato transportation, suggesting a role of human activities in the dispersal of P. infestans in China.

Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

Potato Alternaria leaf blight is one of the economically most important disease in potato production worldwide. A recent study reported a quick method to distinguish main Alternaria pathogens A. tenuissima, A. alternata, and A. solani using partial histone H3 gene sequences. Using this method, our collection of 79 isolates from 8 provinces in China were presumably separated into A. tenussima and A. alternata. But in depth morphological and genetic analysis casted doubt on this identification. Culture morphologies of six presumed A. alternata isolates (PresA_alt) and six presumed A. tenuissima isolates (PresA_ten) were not significantly different. PresA_ten isolates also produced conidia in branched chains which supposed to be A. aternata. Phylogenetic analyses were conducted using internal transcribed spacer region (ITS) and five genes commonly used for species identification including glyceraldehyde-3-phosphate dehydrogenase (GPDH), translation elongation factor 1-alpha (TEF1), ß-tubulin, plasma membrane ATPase (ATPase), and calmodulin genes. The results showed that GPDH and TEF1 sequences of PresA_alt and PresA_ten isolates were identical. The 12 isolates did not cluster by presumed species neither by individual or concatenated sequence comparisons. The phylogeny-trait association analysis confirmed that the two group isolates were undistinguishable by those molecular markers. Analysis of histone H3 gene sequences revealed variable intron sequences between PresA_ten and PresA_alt isolates, but the amino acid sequences were identical. Our results indicate that the previously published method to distinguish Alternaria species based on histone H3 gene sequence variation is inaccurate and that the prevalence of A. tenuissima isolates in China was likely overestimated.

Molecular characterization of two recombinant isolates of telosma mosaic virus infecting Passiflora edulis from Fujian Province in China.

Telosma mosaic virus (TeMV) is an important plant virus causing considerable economic losses to passion fruit (Passiflora edulis) production worldwide, including China. In this study, the complete genome sequence (excluding the poly (A) tail) of two TeMV isolates, Fuzhou and Wuyishan, were determined to be 10,050 and 10,057 nucleotides, respectively. Sequence analysis indicated that Fuzhou and Wuyishan isolates share 78-98% nucleotide and 83-99% amino acid sequence identities with two TeMV isolates of Hanoi and GX, and a proposed new potyvirus, tentatively named PasFru. Phylogenetic analysis indicated that these TeMV isolates and PasFru were clustered into a monophyletic clade with high confidences. This indicated that PasFru and the four TeMV isolates should be considered as one potyvirus species. Two recombination breakpoints were identified within the CI and NIb genes of the Fuzhou isolate, and also within the P1 gene of the Wuyishan isolate. To the best of our knowledge, this is the first report of TeMV recombinants worldwide.

PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies.

Multigene and genomic data sets have become commonplace in the field of phylogenetics, but many existing tools are not designed for such data sets, which often makes the analysis time-consuming and tedious. Here, we present PhyloSuite, a (cross-platform, open-source, stand-alone Python graphical user interface) user-friendly workflow desktop platform dedicated to streamlining molecular sequence data management and evolutionary phylogenetics studies. It uses a plugin-based system that integrates several phylogenetic and bioinformatic tools, thereby streamlining the entire procedure, from data acquisition to phylogenetic tree annotation (in combination with iTOL). It has the following features: (a) point-and-click and drag-and-drop graphical user interface; (b) a workplace to manage and organize molecular sequence data and results of analyses; (c) GenBank entry extraction and comparative statistics; and (d) a phylogenetic workflow with batch processing capability, comprising sequence alignment (mafft and macse), alignment optimization (trimAl, HmmCleaner and Gblocks), data set concatenation, best partitioning scheme and best evolutionary model selection (PartitionFinder and modelfinder), and phylogenetic inference (MrBayes and iq-tree). PhyloSuite is designed for both beginners and experienced researchers, allowing the former to quick-start their way into phylogenetic analysis, and the latter to conduct, store and manage their work in a streamlined way, and spend more time investigating scientific questions instead of wasting it on transferring files from one software program to another.

Molecular Evolutionary Analysis of Potato Virus Y Infecting Potato Based on the VPg Gene.

Potato virus Y (PVY) is an important plant pathogen infecting solanaceous crops, causing significant losses to global potato and tobacco production. Some aspects of the plant pathology and molecular biology of PVY have been studied intensively, but the evolutionary dynamics of this virus are poorly understood. Here, we performed a comprehensive set of rigorous evolutionary analyses using 177 nucleotide sequences of the viral genome linked protein (VPg) gene, which interacts with the plant eukaryotic translation initiation factor 4E (eIF4E). Our Bayesian analysis reveals that the VPg gene of PVY has been evolving at a rate of 5.60 × 10-4 subs/site/year (95% credibility interval 3.35 × 10-4-8.17 × 10-4), which is equivalent to those of other plant-infecting RNA viruses. We identified different evolutionary constraints on the two clades of PVY, clade N and clade O, whose diverge time were estimated at the year 1861 CE (95% credibility interval 1750-1948 CE). We also found that genetic variations were correlated with geographic regions, suggesting that the evolution of this pathogen is strongly affected by geographical associated factors. Taken together, the results of our study have potential implications for the control strategies of PVY.

Molecular characterization of a new potyvirus infecting passion fruit.

A potyvirus (isolate PFV-FJ) infecting passion fruit in China was identified by small-RNA sequencing. The complete genome sequence of PFV-FJ was determined to be 9974 nucleotides, excluding the poly(A) tail. PFV-FJ shares 70-72% nucleotide and 69-74% amino acid sequence identity at the polyprotein level with seven reported potyviruses, but 89% nucleotide and 91% amino acid sequence identity with an unreported potyvirus, tentatively named "passionfruit Vietnam potyvirus" (PVNV-DakNong). This suggests that PFV-FJ and PVNV-DakNong should belong to the same potyvirus species and that PFV-FJ is a new member of the genus Potyvirus. This new potyvirus was tentatively named "passion fruit severe mottle-associated virus".

EasyCodeML: A visual tool for analysis of selection using CodeML.

The genomic signatures of positive selection and evolutionary constraints can be detected by analyses of nucleotide sequences. One of the most widely used programs for this purpose is CodeML, part of the PAML package. Although a number of bioinformatics tools have been developed to facilitate the use of CodeML, these have various limitations. Here, we present a wrapper tool named EasyCodeML that provides a user-friendly graphical interface for using CodeML. EasyCodeML has a custom running mode in which parameters can be adjusted to meet different requirements. It also offers a preset running mode in which an evolutionary analysis pipeline and publication-quality tables can be exported by a single click. EasyCodeML allows visualized, interactive tree labelling, which greatly simplifies the use of the branch, branch-site, and clade models of selection. The program allows comparison of major codon-based models for analyses of selection. EasyCodeML is a stand-alone package that is supported in Windows, Mac, and Linux operating systems, and is freely available at https://github.com/BioEasy/EasyCodeML.

Bayesian phylodynamic analysis reveals the dispersal patterns of tobacco mosaic virus in China.

Tobacco mosaic virus (TMV) is widespread in China and causes considerable economic losses to tobacco production. The molecular epidemiology of this virus is, however, poorly understood. In this study, we sequenced the genomes of 51 TMV isolates from five tobacco-producing regions in China and investigated the dispersal patterns of this virus. Our phylogenetic analysis showed that TMV might have been introduced to China in the early 1900s, probably first to southwest China. However, TMV then moved to the north of the country, where it expanded. The north became the main seeding region for the subsequent movements of the virus within China. The north-to-south movement of TMV coincides with a shift of major tobacco-producing areas from north to south in this century, suggesting a link between human activities and the dispersal of TMV in China.

Europe was a hub for the global spread of potato virus S in the 19th century.

Potato virus S (PVS) is a major plant pathogen that causes considerable losses in global potato production. Knowledge of the evolutionary history and spatio-temporal dynamics of PVS is vital for developing sustainable management schemes. In this study, we investigated the phylodynamics of the virus by analysing 103 nucleotide sequences of the coat protein gene, sampled between 1985 and 2014. Our Bayesian phylogenetic analyses showed that PVS has been evolving at a rate of 3.32 × 10-4 substitutions/site/year (95% credibility interval 1.33 × 10-4-5.58 × 10-4). We dated the crown group to the year 1325 CE (95% credibility interval 762-1743 CE). Our phylogeographic analyses pointed to viral origins in South America and identified multiple migration pathways between Europe and other regions, suggesting that Europe has been a major hub for PVS transmission. The results of our study have potential implications for developing effective strategies for the control of this pathogen.

Rapid evolutionary dynamics of pepper mild mottle virus.

Pepper mild mottle virus (PMMoV) is one of the most destructive pathogens of pepper crops and has major impacts on global crop yields. Some aspects of the molecular biology of PMMoV have been studied intensively, but estimates of its evolutionary rate have shown considerable variation. We investigated the phylodynamics of PMMoV by analysing 171 nucleotide sequences of the coat protein gene, sampled between 1980 and 2016. Our Bayesian phylogenetic analyses, using the structured coalescent, dated the crown group to 1949 (95% credibility interval 1935-1962). We reveal that PMMoV has been evolving at a rate of 9.363 × 10-4 substitutions/site/year (95% credibility interval 7.362 × 10-4-1.138 × 10-3). This is similar to evolutionary rates estimated for animal RNA viruses, indicating that PMMoV has been undergoing rapid evolutionary dynamics.

Genetic diversity and molecular evolution of Ornithogalum mosaic virus based on the coat protein gene sequence.

Ornithogalum mosaic virus (OrMV) has a wide host range and affects the production of a variety of ornamentals. In this study, the coat protein (CP) gene of OrMVwas used to investigate the molecular mechanisms underlying the evolution of this virus. The 36 OrMV isolates fell into two groups which have significant subpopulation differentiation with an FST value of 0.470. One isolate was identified as a recombinant and the other 35 recombination-free isolates could be divided into two major clades under different evolutionary constraints with dN/dS values of 0.055 and 0.028, respectively, indicating a role of purifying selection in the differentiation of OrMV. In addition, the results from analysis of molecular variance (AMOVA) indicated that the effect of host species on the genetic divergence of OrMV is greater than that of geography. Furthermore, OrMV isolates from the genera Ornithogalum, Lachenalia and Diuri tended to group together, indicating that OrMV diversification was maintained, in part, by host-driven adaptation.

The first complete genome sequence of narcissus latent virus from Narcissus.

The complete sequence of a narcissus virus isolated from the Netherlands (Narv-NL) was determined to be 8172 nucleotides in length with an open reading frame encoding for 2624 amino acids. Phylogenetic analysis indicates that Narv-NL is clustered with high confidence among representative members from the genus Macluravirus, including artichoke latent virus (ArLV) and Chinese yam necrotic mosaic virus (CYNMV). Sequence analyses indicated Narv-NL shares 67%-69% nucleotide and 51%-68% amino acid sequence identity with ArLV and CYNMV either in the complete ORF or the coat protein (CP) gene, whereas it had 81%-99 % nucleotide and 80%-99 % amino acid sequence identity with the corresponding CP sequences of narcissus latent virus (NLV) isolates, suggesting that Narv-NL is a member of NLV. To our knowledge, this is the first report of the complete sequence of a NLV isolate.