Whole CBSV/UCBSV genomes (nucleotides) were analyses using a Bayesian analyses were conducted using ExaBayes version 1.4.1; [35] and were run in parallel across 384 nodes on the Magnus supercomputer (located at the Pawsey Centre, Western Australia).

<p>Analyses were run for 1 million generations with sampling every 500 generations. Convergence was evaluated using Tracer. Each analysis consisted of four independent runs, each utilising four coupled Markov chains.</p

Recombination patterns observed from analysis involving the 12 new sequences along with those retrieved from GenBank; a) Eight firm recombination events identified amongst the new Cassava brown streak virus sequences when compared with all those available on GenBank; and b) Four firm recombination events identified amongst the new Ugandan cassava brown streak virus sequences when compared with all those available on GenBank.

<p>Recombination patterns observed from analysis involving the 12 new sequences along with those retrieved from GenBank; a) Eight firm recombination events identified amongst the new Cassava brown streak virus sequences when compared with all those available on GenBank; and b) Four firm recombination events identified amongst the new Ugandan cassava brown streak virus sequences when compared with all those available on GenBank.</p

Cassava brown streak disease symptoms seen on infected cassava plants collected from the field; a) Mafia district, feathery patterns with chlorotic blotches along the margins of secondary, tertiary and main veins; b) Tanga district, severe yellow chlorosis on leaves; c) Nyasa district, moderate leaf chlorosis and d) Serengeti district, moderate chlorosis, mottling and chlorotic blotches.

<p>Cassava brown streak disease symptoms seen on infected cassava plants collected from the field; a) Mafia district, feathery patterns with chlorotic blotches along the margins of secondary, tertiary and main veins; b) Tanga district, severe yellow chlorosis on leaves; c) Nyasa district, moderate leaf chlorosis and d) Serengeti district, moderate chlorosis, mottling and chlorotic blotches.</p

The species delimitation plugin generates: Intra Dist: average pairwise tree distance among members of a predefined clade, Inter Dist: average pairwise tree distance between members of the group of interest and its sister taxa (K2Pdistance), Intra/Inter: The ratio of Intra Dist to Inter Dist, P ID(Liberal): mean probability, with a 95% confidence interval (CI) for a prediction of making a correct identification of an unknown specimen being sister to or within the group of interest, P ID (Strict): mean probability, with a 95% confidence interval (CI) for a prediction of making a correct identification of an unknown specimen being found only in the group of interest [56], Av(MRCA): mean distance between the most recent common ancestor of the species and its members, P(Randomly Distinct): probability that a clade has the observed degree of distinctiveness [38], Clade Support: Bayesian posterior probability (PP), and Rosenberg’s P_AB: Reciprocal monophyly.

<p>* Indicates significant species delimitation.</p><p>The species delimitation plugin generates: Intra Dist: average pairwise tree distance among members of a predefined clade, Inter Dist: average pairwise tree distance between members of the group of interest and its sister taxa (K2Pdistance), Intra/Inter: The ratio of Intra Dist to Inter Dist, P ID(Liberal): mean probability, with a 95% confidence interval (CI) for a prediction of making a correct identification of an unknown specimen being sister to or within the group of interest, P ID (Strict): mean probability, with a 95% confidence interval (CI) for a prediction of making a correct identification of an unknown specimen being found only in the group of interest [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.ref056" target="_blank">56</a>], Av(MRCA): mean distance between the most recent common ancestor of the species and its members, P(Randomly Distinct): probability that a clade has the observed degree of distinctiveness [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.ref038" target="_blank">38</a>], Clade Support: Bayesian posterior probability (PP), and Rosenberg’s P_AB: Reciprocal monophyly.</p

Recombination events in the coding regions of <i>Ugandan cassava brown streak virus</i> (UCBSV) genomes when analyzed with UCBSV and <i>Cassava brown streak virus</i> genomes.

<p>Recombination events in the coding regions of <i>Ugandan cassava brown streak virus</i> (UCBSV) genomes when analyzed with UCBSV and <i>Cassava brown streak virus</i> genomes.</p

DensiTree [40] is a visualization tool that displays all trees encountered as the Markov Chains samples the large tree space.

<p>Two-thousand trees are depicted in the Densitree and there is clearly more uncertainty in the CBSV relationships as visualized by the loose clustering of the lines associated with the CBSV cluster. Conversely, there is tight clustering of the UCBSV samples indicating more confidence in the relationships within the species.</p

Recombination events in the coding regions of <i>Cassava brown streak virus</i> (CBSV) genomes when analyzed with CBSV and <i>Ugandan cassava brown streak virus</i> genomes collected in Tanzania.

<p>Recombination events in the coding regions of <i>Cassava brown streak virus</i> (CBSV) genomes when analyzed with CBSV and <i>Ugandan cassava brown streak virus</i> genomes collected in Tanzania.</p

Comparison of all phylogenetic trees showing and the complete genome tree (NA)-Fig 2 is the reference.

<p>(+) Indicates that the clade contains the same species as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.g002" target="_blank">Fig 2</a> and occurs in the same location on the tree. (-) Indicates that the clade contains different species and/or is in a different location on the tree when compared to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.g002" target="_blank">Fig 2</a>.</p

Maps of Tanzania showing the distributions of samples and sequences used in this study.

<p>a) Foliar Cassava Brown Streak Disease (CBSD) severity score 1–5. Leaf symptom severity was scored on 3-month-old plants using a five point scale where 1 = no visible CBSD symptoms, 2 = mild foliar symptoms on some leaves, 3 = pronounced foliar symptoms but no die-back, 4 = pronounced foliar symptoms which might include slight die back of terminal branches, and 5 = severe foliar symptoms and plant die-back [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139321#pone.0139321.ref018" target="_blank">18</a>]. b) Geographical distribution of new cassava brown streak virus isolates, CBSVs and UCBSVs used in the present study. Locational names abbreviation in virus isolate names are: Nya = Nyasa; MAF = Mafia; Tan = Tanga, and Ser = Serengeti.</p

Unusual occurrence of a DAG motif in the Ipomovirus <i>Cassava brown streak virus</i> and implications for its vector transmission

<div><p>Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating <i>Ipomoviruses</i> that cause cassava brown streak disease (CBSD); <i>Cassava brown streak virus</i> (CBSV) and <i>Ugandan cassava brown streak virus</i> (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used <i>Bemisia tabaci</i> (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52–54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: <i>Squash vein yellowing virus</i> (SqVYV), <i>Coccinia mottle virus</i> (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at <i>Ipomovirus</i> genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.</p></div