# Bayesian phylogenetic and recombination analyses of plum pox virus provide a refined vision of its evolutionary history

F Palmisano, S Kawakubo, M Chiumenti, P Leonetti, V Pantaleo, T Candresse, A Minafra

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F Palmisano, S Kawakubo, M Chiumenti, P Leonetti, V Pantaleo, et al., Bayesian phylogenetic and recombination analyses of plum pox virus provide a refined vision of its evolutionary history. Virology Journal, 2025, 22, 10.1186/s12985-025-02892-7. doi: 10.55026/28

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# Bayesian phylogenetic and recombination analyses of plum pox virus provide a refined vision of its evolutionary history

F. Palmisano<sup>1,4†</sup>, S. Kawakubo<sup>2,5†</sup>, M. Chiumenti<sup>1</sup>, P. Leonetti<sup>1</sup>, V. Pantaleo<sup>1*</sup>, T. Candresse<sup>3</sup> and A. Minafra<sup>1*</sup>

**Abstract**

**Background** The discovery of a plum tree isolate of plum pox virus (PPV, *Potyvirus plumpoxi*), done in Eastern Albania in 2011 in the frame of an EU-funded survey, which represents a divergent strain named PPV-An, proved to be original and informative for the unraveling of PPV evolutionary history.

**Methods** Maximum likelihood and Bayesian phylogenetic methods applied on full-length genomes or selected regions analyzed the affinities of the PPV-An with other PPV strains. Potential recombination events were also evaluated. A refined timeline of PPV evolutionary history integrating recombination events, strains migration and ancestral host state is proposed.

**Results** Altogether, the analyses confirm previous hypotheses that PPV-An corresponds to an ancestral, non-recombinant PPV strain. PPV-An likely served as the origin of the PPV-M and T strains through recombination with isolate(s) of the D strain. Molecular clock analyses dated the most recent common ancestor (TMRCA) of PPV at 4546 years ago and phylogeny separated the main PPV strains from the cherry-adapted strains around 3100 years ago. Meanwhile, the recombination events that gave rise to the M and T strains are estimated to have occurred in the early sixteenth century of the common era (CE).

**Conclusions** The characterization of the PPV-An strain enabled a comprehensive phylogenetic analysis of PPV. PPV-An is confirmed to be the previously unidentified progenitor, which, together with PPV-D led through recombination to the emergence of the currently prevalent and evolutionary successful recombinant strains of European origin (e.g., M, Rec, and T). The low representation of PPV-An in current PPV populations is likely the consequence of a population replacement phenomenon possibly linked to a higher fitness of the recombinant strains deriving from it. These results highlight the PPV-An strain as a key player in PPV evolutionary history and consolidate PPV as one of the promising models to study host-adaptive evolution processes and phylogeography among the most damaging viruses of agricultural systems.

**Keywords** Sharka disease, Plum pox virus, Strain dynamics, Phylogeographic inference, Recombination, Ancestor

<footnote>†F. Palmisano and S. Kawakubo contributed equally to this work.</footnote>

<footnote>*Correspondence:
V. Pantaleo
vitantonio.pantaleo@cnr.it
A. Minafra
angelantonio.minafra@cnr.it
Full list of author information is available at the end of the article</footnote>
## Background

*Potyvirus plumpoxi* (plum pox virus, PPV) is a member of the genus *Potyvirus*, family *Potyviridae*. Like other potyviruses, PPV has a single-stranded positive-sense genomic RNA of about 10 kb which encodes a single large open reading frame (ORF). Translation of this ORF generates a polyprotein precursor of ~350 kDa that is in turn processed, giving rise to processing intermediates and to 10 final protein products [1]. Potyvirids also encode a second, small ORF named PIPO, via a ribosomal frameshifting within the P3 cistron. PPV is the agent of the Sharka disease, the most devastating disease of stone fruit trees worldwide [2–7]. Accordingly, PPV is considered as either a quarantine pathogen or a regulated non-quarantine pathogen in a wide range of countries [4, 5, 8].

PPV is transmitted by several species of aphids [9], which acquire the virus when probing infected plants and then transfer it in a non-persistent manner to healthy plants. Once a tree is infected, it can exhibit various symptoms including discolored leaf arabesques and rings, leaf rugosity (wrinkling), and fruit deformations such as ringspots or necrotic spots. Severe fruit drops may occur in the most susceptible varieties. In addition, PPV is transmitted through all vegetative propagation techniques, such as grafting, making the trade of *Prunus spp.* propagation material responsible for its long-range propagation, including intercontinental spread.

The genomes of a broad range of PPV isolates have been completely sequenced [10–16] and PPV has been studied from both phylogenetic and evolutionary perspectives. Researchers have used molecular techniques to analyze the genetic diversity of PPV isolates collected from different geographic areas and host species. These studies provided insights into the evolutionary relationships among different strains of the virus and helped to trace the spread of PPV populations [17–21]. So far, 10 strains of PPV have been recognized and named. The three major strains, which show the broader geographic distribution, are PPV-D (Dideron; [22]), PPV-M (Marcus, [22] and PPV-Rec (Recombinant). The other identified strains show more limited geographic distributions, including the PPV-EA (El Amar; [10, 23]) identified in Egypt, a few other strains with a localized and sporadic presence such as PPV-W (Winona; [12]), PPV-T (Turkey; [13]), and PPV-An (Ancestor; [24]). At least 3 different strains are able to naturally infect sweet and sour cherry PPV-C (Cherry, [11]), PPV-CR (Cherry Russia, [25]), PPV-CV (Cherry Volga, [14]), while several phylogroups of cherry-adapted isolates (namely SC, TAT and Y) that may represent further strains have been recently described in the South-Eastern part of Russia [15].

Understanding the phylogenetic relationships between PPV strains is crucial not only for addressing the evolutionary history of PPV but also for developing and implementing effective control strategies, such as deploying resistant cultivars or designing diagnostic tools for early strain-specific detection and spread prevention. Moreover, studying the evolutionary dynamics of PPV can provide insights into how the virus evolves in response to selective pressures, such as host resistance mechanisms or changes in vector populations [26]. The wealth of PPV genetic information available from public repositories and associated metadata makes PPV an interesting model for phylogeographic and evolutionary studies [20, 27, 28]. It is widely accepted that recombination has been a major driving force in PPV evolution. For instance, the PPV-Rec strain originated from recombination between PPV-M and PPV-D, with a breakpoint in the NIb gene 3'region [29]; due to its typical PPV-M coat protein, it was long misidentified as PPV-M [7]. Similarly, a Canadian PPV-W isolate was identified as a complex recombinant involving PPV-W, PPV-M, and PPV-D [30]. Moreover, although the P1 protein is the least conserved among potyviral proteins and is believed to be involved in potyviruses host adaptation [31], the 5' part of the genome shows high homology among the PPV-D, -M, -T, and -Rec strains but not with other strains, suggesting these four strains are linked by (an) ancestral recombination event(s). The precise recombination history linking these strains is complex to unravel, and two tentative recombination breakpoints have been proposed in the HC-Pro and the P3 genes [17]. Two scenarios for the evolutionary history of PPV have been proposed, in which either PPV-D or PPV-M (and PPV-T) would be (a) recombinant strain(s), while the other would have contributed as one of the parents involved in the corresponding recombination event [17].

In both scenarios, the existence of an ancestral, non-recombined form of PPV-D or PPV-M was postulated. Partial genome sequencing of PPV isolates collected during field surveys performed in Albania in 2011 demonstrated the presence of several PPV isolates [32](F. Palmisano and S. Dallot, unpublished) and allowed the identification of an unusual isolate named AL-11pl, which was characterized by a divergent 5'genomic region while the rest of its genome is more typical of already known isolates of the PPV-M and -T strains. These features fit with the properties hypothesized by Glasa & Candresse [17] for a putative ancestral strain which could have contributed, in different recombination events with PPV-D, to the emergence of the -M and -T strains [7]. The discovery and the characterization of the AL-11pl isolate thus provide support for one of the two alternative evolutionary scenarios proposed by Glasa & Candresse [17] and
lead to the naming of the corresponding strain as PPV-An (Ancestral of Marcus strain) [24]. Although an initial analysis found faint evidence linking PPV-T to PPV-An [19], a global phylogenetic analysis confirmed a strict association between PPV-An and the PPV-T clade [18]. For the first time, these latter authors [18] proposed a recombination events-based global PPV phylogeny on a dataset of 206 isolates, and estimated the age of the most common recent ancestor at about 800 years before common era through sub-tree comparison method. Recent advances in the study of the phylogeny and molecular evolution of potyviruses [33] have integrated Bayesian phylogenetic analysis and dating approaches, as demonstrated in research on PVY [34] and TuMV [35, 36]. Genome characterization and Bayesian phylogenetic inference of georeferenced isolates of these viruses have facilitated the reconstruction of their spread and evolutionary history [37, 38].

In the present study, we used Maximum Likelihood phylogeny and Bayesian approaches for the analysis of molecular evolution and timing of PPV strains differentiation [39–42]. For this, we used the complete AL-11pl sequence and those of an additional 539 PPV isolates to tentatively date the appearance of the PPV-An-derived lineage. From these elaborations, it was possible to propose a hypothetical scenario about the geographical origin and host ancestral state of PPV and for the further spatio-temporal spread of its strains.

## Methods

### Recovery from public databases of PPV sequences associated with their metadata and reconstruction of their phylogeny

A total of 609 full length PPV genomes were downloaded on 07 November 2023 from the NCBI Virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/). Sequences showing frameshifts or interruptions of the polyprotein were excluded from further analysis. Metadata (name of isolate, sampling year, host and country)

associated with the remaining sequences were downloaded and manually curated. A total of 539 sequences for which complete metadata were available, were kept for subsequent analyses (Additional Table 1). A multiple alignment of these full-length genomic sequences was obtained using MAFFT [43]. The aligned complete genomic sequences were further manually checked in Geneious v.2024.0.7 to identify the ends of the large ORF encoding the polyprotein and the correct genome termini. Pairwise identity percentages were calculated from the aligned positions.

In addition to the phylogenetic analysis run on the full-length genome alignment, three distinct genomic regions were selected, avoiding the breakpoints of known recombination events already described and confirmed here (see below and Fig. 2D) [7, 17]: (i) a 5' terminal region (nt 36–1400, the numbering used throughout follows the consensus alignment using all 539 isolates); (ii) an internal 5' fragment (nt 1500–2600); (iii) a 4 kilobases central region (nt 3500–7500). The best fitting substitution models for the full-length genome and various partial sequences alignments were investigated using MEGA X [44] and ModelFinder implemented in IQ-tree (version 2.3.6 [45];). The substitution model suggested by this latter program was finally applied to the test. Maximum likelihood (ML) phylogenies were then inferred using the IQ-Tree software. Bootstrap values were calculated using 1,000 replicates. Trees were finally visualized in the Interactive Tree of Life (iTOL) [46].

### Recombination analysis of genomic sequences

To reconstruct the evolutionary history of PPV, and to understand the possible origin of the genomic fragments and the role played by recombination in such evolution among the strains, a search for potential recombination breakpoints and the identification of the putative parents was performed using the RDP4 program (version 4.10.1, default program settings) [47] and the above described full-length genomes multiple sequence alignment. Only

<table><caption>**Table 1** Percentages of identity with PPV-An (isolate AL-11pl) in different genome regions. Values are derived from pairwise identity matrices obtained from a MAFFT multiple alignment of full-length genomes of 539 PPV isolates. Metadata (year, host and country of isolation) for each reported isolate are provided</caption><thead><tr><th>Region (nucleotide position)</th><th>MAFFT alignment</th><th>Identity (%)</th><th>Strain</th><th>Metadata of strains/sequence</th></tr></thead><tbody><tr><td rowspan="2">1-1584</td><td>max</td><td>81.7</td><td>T</td><td>ON745776|P13_ANK|Turkey|Prunus_cerasus|2017</td></tr><tr><td>min</td><td>73.2</td><td>CV</td><td>MF447179|Tat_2|Russia|Prunus_cerasus|2015</td></tr><tr><td rowspan="2">1585-2758</td><td>max</td><td>96.3</td><td>T</td><td>MF346246|AnKuAp8|Turkey|Prunus_armeniaca|2014</td></tr><tr><td>min</td><td>76.8</td><td>TAT</td><td>OK562686|TAT_85|Russia|Prunus_cerasus|2018</td></tr><tr><td rowspan="2">2759-7532</td><td>max</td><td>96.4</td><td>M</td><td>LC494682|Y3|Japan|Prunus_mume|2016</td></tr><tr><td>min</td><td>77.6</td><td>W</td><td>HQ670746|LV_141pl|Latvia|Prunus_domestica|2010</td></tr></tbody></table>
events detected by at least 4 methods and with corrected
$p$-values < $10^{-6}$ were considered as reliable. The identi-
fied breakpoints were further manually examined, and
BLASTN searches were used to verify the parent/donor
strain assignment and their respective sequence homol-
ogy levels.

Ancestral states reconstruction through Bayesian
phylogeny

The existence of a temporal signal of PPV evolution
in the 539 full-length genomic sequences dataset was
assessed using the programs TempEst (version 1.5.3; [48])
and TreeTime (version 0.11.4; [49]).

For Bayesian phylogenetic analysis, two datasets were
used. The first corresponds to the central genomic region
(nt 3500–7500) aligned for the 539 isolates representing
all strains in our original dataset. This region was shown
to be free of recombination events for the isolates used
and will be hereafter referred to as the 4 K region. The
second one corresponds to the 5' genome fragment (nt
36–1406) from the same PPV full-length genome align-
ment. The package bModelTest [50] was used to assess
the site model and associated substitution model. Mar-
ginal likelihood estimation (MLE) through Path sam-
pling/Stepping-stone sampling (PS/SS) analysis [51] was
run on BEAST to compare the fit of the relaxed clock
model with a strict clock model in the above-mentioned
sequence datasets. Based on the available metadata (see
paragraph above), for each PPV sequence available dis-
crete location (country of origin) and host of origin
states were assigned. A symmetric substitution model
was applied for each discrete trait (host and country)
and social networks were inferred by Bayesian stochas-
tic search variable selection (BSSVS). Ancestral states
were reconstructed for all the considered partitions. The
Markov Chain Monte Carlo (MCMC) method in the
BEAST package (v10.5.0; [52]), along with BEAGLE, was
inferred with the best fit substitution model (GTR+G+I)
suggested by bModelTest for the two independent data-
sets described above. In the case of the 4 K region, 13
runs for a total of 1300 million MCMC steps were carried
out, while in the case of the 5' region 3 runs including 800
million steps, were merged using LogCombiner (v10.5.0).
The Tracer software (v 1.7.2) was used to confirm that
all estimated parameters yielded effective sampling sizes
(ESS) greater than 200 and that 10% of the total chain
length had been burned-in to reduce the influence of the
initial value. The final Bayesian maximum clade credibil-
ity (MCC) tree was generated by TreeAnnotator (v10.5.0)
and visualized in Figtree (v 1.4.4). A graphical elaboration
of the final MCC trees was obtained in RStudio using the
packages ggtree and treeio [53, 54]. The resulting Bayes-
ian diffusion states in space and time were calculated

with the SPREAD application (version 1.0.7; [55]). The
ancestral trait for the host was reconstructed at the root
and the oldest nodes for the same MCC trees.

To further confirm the molecular clock signals obtained
through the previously described BEAST analysis, a syn-
chronous BEAST elaboration for the two datasets (sim-
ulating the sampling dates as done all at the same time,
i.e. 2017.5) was run with the same parameters described
above. Then, date-randomization test was performed
with 10 replicates whose sampling date were randomly
permutated in the TipDating Beast package. We assumed
the presence of temporal structure when the 95% cred-
ible interval of the rate estimate from the original dataset
did not overlap with the 95% credible interval of any of
the rate estimates from the date-randomized replicates.

Finally, to understand if the prevalence of PPV-D
sequences present in the datasets could have biased the
Bayesian calculation of substitution rates and TRMCAs,
an additional analysis was performed with the very same
parameters described above for the 5' and 4 k regions,
selecting only PPV-D non recombinant and PPV-Rec
strains, for a total of 291 sequences.

Results

Relevant molecular and serological features of PPV-An

The AL-11pl isolate (GenBank HF674399), which typifies
the PPV-An strain and will be hereafter indicated as An,
was identified in a domestic plum tree in Eastern Alba-
nia. Serologically, PPV-An reacted to the PPV-M-specific
monoclonal antibody (MAb) AL (not shown). Remark-
ably, it also tested positive with the PPV-D-specific MAb
4DG5, an unusual behaviour previously reported for
some PPV-T isolates [56]. The complete genome of the
PPV-An isolate is 9,786 nucleotides long, excluding the
3' terminal polyA tail, and has a GC content of 43.8%.
The genomic organization is typical of members of genus
*Potyvirus*, and identical to that of other PPV isolates. A
start codon (AUG) is present at positions 147–149, and
an amber stop codon at positions 9567–9569, resulting
in a single open reading frame (ORF) of 9420 nt/3140
amino acids. In addition, the PIPO ORF [57] putatively
encoding a 12 kDa protein was identified in the P3 coding
region as a +2 frameshift sequence starting at nucleotide
position 2906.

Sequence alignments show a conservation of the nine
polyprotein cleavage sites as compared to PPV-M iso-
lates, with the exception of mutations observed in the
NIa-VPg/NIa-Pro cleavage site (EEVGHE/S in PPV-An
and DEVDHE/S in PPV-M isolates) and NIa-Pro/NIb site
(EFVHNQ/S vs. EFVYNQ/S). All conserved motifs typi-
cal of potyviruses were also identified at their expected
locations, including the KITC [58], PTK [59] and DAG
motifs associated with aphid transmission.
Percentages of nucleotide pairwise identity calculated between PPV-An and all other PPV strains – expressed as their maximal and minimal values in various genomic regions selected from the full-length genome alignment are given in Table 1. The complete genome sequence comparisons indicate that the closest strain to PPV-An is PPV-T, with an overall nucleotide identity of 93.5%. Nevertheless, while PPV-An is closely related to isolates of the T and M strains at the whole genome level, it shows a much lower identity level (74–77%) with these strains for the 5' non-coding region (5'NCR) and the P1 gene (81.7%, from start codon up to nt 1584). This dissimilarity pattern extends to the HC-Pro gene (nt 1585–2758) in the case of the M strain (82% nt sequence identity) but it is no longer observed in the case of the T strain (96.3% identity; Table 1). Nucleotide identity levels with both T and M strains for all the 3' downstream parts of the genome are higher than 95%. This unusual identity pattern observed for the various regions of the PPV-An, strongly points to its possible implication in recombination events.

## Analysis of recombination events involving PPV-An as parental ancestor

The multiple alignment of 539 full-length genomic sequences of PPV isolates was analysed for recombination events using RDP4. A number of putative recombination events were identified by the program, but most of them involved just one or a few isolates and were detected by only three or less of the methods, with statistically non-significant *p*-values. However, two recombination events which involve strains sharing a large genomic portion with PPV-An were identified by a strong signature and are discussed in detail here (Additional Table 2). A first statistically significant recombination event (i.e. lowest *p*-value $1.77 \times 10^{-73}$) identifies the fragment at nt positions 1561–2735 in the alignment (with 99% confidence intervals [CI 99%] for the breakpoints at 903–1598 and 2678–2765) and affects 130 PPV-M isolates (Fig. 1A; event #4, Additional Table 2). This event involves a putative major parent belonging to the PPV-T strain (GenBank MF346274), and a minor parent belonging to the PPV-D strain (GenBank KR006730). Given the RDP4 output, strain T isolates are interpreted by the program as non-recombinant major donors of a backbone which received the insertion of the heterologous fragment from a strain D minor parent, thus leading to the current PPV-M isolates.

The other relevant recombination event identified, with a breakpoint at nt 2691 of the PPV-An sequence (CI 99%: 2665–2816), involves a PPV-M isolate as the major parent providing the entire 3' genome part (from nt 2691 up to 3' end) and an unknown minor parent providing

the 5'genome portion (Fig. 1A; event #3, Additional Table 2). This recombination event is consistently supported by six methods (i.e. RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan) with the lowest *p*-value of $2.23 \times 10^{-115}$. The breakpoint of this predicted recombination event is in very close proximity to the one (at position 2814) hypothesized by Glasa & Candresse [17] for a recombination event between PPV-D and a putative ancestral isolate leading to the emergence of the PPV-M strain. However, when the 3' end genomic region of PPV-An (nt 3500–9700) was used as a query for a BLASTN interrogation of GenBank, the closest isolate to PPV-An in that genomic region was PPV-T KrPnPl345 (GenBank MF346272), sharing 96.4% of nt identity. For the 5' terminal region (1–1561), an unknown minor parent was postulated in the RDP4 output. A BLASTN analysis indicated that the isolate most similar to PPV-An in that region is P93 ANK (GenBank ON745778), another PPV-T isolate, but its identity with PPV-An is only of 85,3%.

The #3 and #4 (Additional Table 2) events identified here are respectively similar to events X2 and X3 identified by [18], although a reasoning based on phylogenetic analysis led them to conclude that the T strain was derived from a PPV-M strain parent, and not the reverse as predicted by our RDP analysis. However, the potential scenario described by these authors has two weaknesses. First, considering either PPV-M or PPV-T as a non-recombined strain parent to PPV-An fails to provide an explanation for the low divergence in the P1 protein between these two strains and PPV-D strain, despite the fact that P1 is very generally the most divergent potyviral protein. Second, this scenario is not parsimonious since it needs to postulate the existence of a further unknown minor parent providing the 5'terminal portion of PPV-An in the recombination event #3. In contrast, the scenario proposed by [17] and further discussed by [7], which identifies PPV-An and PPV-D as ancestral non recombinant parents and PPV-M and PPV-T as the derived recombinant progeny, is more parsimonious (it does not postulate the existence of another unknown parent) and readily provides an explanation for the high P1 homology observed between PPV-D and M and T strains (Fig. 1B, Hypothesis 2).

## Maximum likelihood phylogenetic analyses on full length sequences and partial genomic fragments

The maximum likelihood (ML) phylogenetic analysis performed on the full-length genome dataset showed a striking position of PPV-An as a long branch linked to the PPV-T clade (Additional Fig. 1). This analysis shows very clearly a separation between the clades of isolates belonging to the European and Mediterranean strains
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 1** Schematic representation of the RDP4 output for some recombination events and hypothesis of recombination involving PPV-An strain. A Schematic representation of PPV genomic organization, where the open box represents the translated ORFs and the functional polyprotein fragments are named. B In the hypothesis 1 frame, the representation only pictures two independent recombination events as identified by RDP4 in terms of parental PPV strains and recombined regions and is not related to any phylogenetic assumption. Here, PPV-An is considered as recombinant. Event #3 depicts the PPV-An isolate, AL-11pl, as originated by recombination between an unknown minor parent providing the 5'-end fragment (nt positions 1–2691) and a major parent belonging to M strain and providing the rest of the genome. Event #4 depicts the origin of the M strain isolates through the insertion of a fragment (nt positions 1561–2735) from a D isolate in the backbone of a T isolate (see Additional Table 2). C In the hypothesis 2 frame, the scenario in which An is considered non-recombinant and a parent of M and T strains, when recombining with PPV-D, is illustrated. The different color codes of the strains derive from the different roles as parent or recombinant they cover in the different events</figcaption></figure>

(PPV-M, -T, -An, -D and -EA) from those corresponding to PPV-W and the cherry-adapted strains. As an alternative to the exclusion of the large number of recombinant isolates (PPV-M, T and Rec strains) from the dataset to perform an evaluation of the phylogenetic signals (therefore losing useful information), we decided to use only genome portions known to be free of recombination breakpoints. The best substitution model obtained with ModelFinder for the full-length genomes and the

three partial genomic datasets was GTR+F+I+G4, and it was thoroughly applied for the further phylogenetic calculations.

A phylogenetic analysis was performed using a 5'genomic fragment (nucleotide positions 36 to 1400), which ends before the border of the first recombination breakpoint suggested by [17] and essentially overlaps with the 5'border of the recombination event #4 discussed above. The resulting ML cladogram showed
a tight clustering of all PPV-D, PPV-M, and PPV-T isolates (61.2% bootstrap), which were separated from a small clade containing the PPV-EA and PPV-An isolates (59.2% bootstrap) (Fig. 2A). In the ML tree reconstructed using the region between the first and second identified recombination breakpoints (nt positions 1500–2600, according to [17] and which spans the C-terminal half of HC-Pro and the beginning of the P3 gene (Fig. 2B), PPV-An now forms a loose cluster (41.4% bootstrap) with the PPV-T isolates.

Lastly when a large, non-recombined internal genome fragment (nt 3500–7500, referred as the 4 K region) was used for ML phylogeny, PPV is split into five phylogenetic groups (Fig. 2C) with PPV-An clearly flanking, as a single long branch, the PPV-M and PPV-T clade (65.4% bootstrap).

In this tree, a single isolate (SK-111pl, belonging to M strain, GenBank HF585099) has an anomalous position as basal tip at the node which originated all the An-derived strains. This position could most likely be due to the peculiar recombination history of this isolate, which concerns, according the RDP4 analysis, a recombination event involving a small region (from nt 3264 to 4010; Additional Table 2, event #5) at the beginning of the 4 K region. Overall, the incongruent topologies in the different trees when it comes to the PPV-D, Rec, M, T isolates versus the An one, support the recombination analysis and confirms that recombination events have contributed in a major way to PPV evolutionary history of the Euro-Mediterranean (Euro-Med) PPV strains. Figure 2D resumes the current view of recombination events along the PPV genome which characterize the single strains.

<figure><img src="image_6.png" /></figure>

**Fig. 2** Maximum likelihood phylogenetic trees reconstructed using different genome regions of 539 PPV isolates. Strains or phylogroups are indicated as PPV-T (T), PPV-An (An), PPV-M (M), PPV-EA (EA), PPV-W (W), PPV-C (C), PPV-Y (Y), PPV-CV (CV), PPV-SC (SC), PPV (TAT), PPV-CR (CR), PPV-D (D) and PPV-Rec (Rec). Colored spots (when present) correspond to the Prunus species from which any isolate was obtained (see the legend of Additional Fig. 1). All the strains belonging to the same clade and sharing similar sequence identity in the genomic portion under evaluation, are grouped by the same color background. The distance bar is also shown under each tree. **A** Tree for the 5' region (nt position 36–1400). **B** Tree for an internal fragment corresponding to nt positions 1500–2600. **C** Tree for the 4 K genomic fragment (nt position 3500–7500). **D** Color-coded schematic representation of the genome of the main PPV strains. Different colors represent the tentative strain origin of the putative recombined fragments. The genome organization of PPV is shown at the top, where the open box represents the translated ORFs and the functional polyprotein fragments are named. The bootstrap values at the main nodes are not reported in the pictures to improve visualization, since most of them are > 60
## PPV-An-derived strains diversification and Bayesian dating in the global PPV evolution and spread

The presence of a temporal signal was first investigated on the global PPV phylogeny to determine whether analyzing the evolution of existing lineages could provide a reliable estimate for the time to a common ancestor between An and the other PPV lineages. The TempEst software [48] run on the full-length PPV genomes, recovered a TMRCA intercept at 3604 years ago, while the root-to-tip regression using TreeTime put the TMRCA inferred from the same sequence alignment at 4499 years ago. The quantitative metrics obtained with TempTest are reported in Additional Table 3.

This preliminary dating analysis for a retrieval of a temporal signal could incur a bias because of the inclusion of recombinant sequences in the dataset. In fact, according to our evidence of PPV recombination history (Fig. 2D) only PPV-D, PPV-An, PPV-EA, most of PPV-W and the cherry-adapted strains are considered non-recombinants, so that a large proportion of the isolates included in the dataset are recombinants. To overcome this problem, Bayesian datation and phylogeographic diffusion analysis were separately performed on two non-recombinant genome regions described above: the 4 K central region and the shorter 5' fragment. First, to evaluate the temporal structure in the dataset, the date-randomization test was performed. The 95% credible intervals of inferred substitution rate for both datasets did not overlap with any of the date-randomized replicates, indicating the presence of temporal signal in our dataset (Additional Fig. 3, A and B). Moreover, the presence (strict clock model) and absence (uncorrelated relaxed clock model) of a molecular clock signal were also tested using Bayesian statistics. MLE calculated for the two hypothesized models through PS/SS analysis yielded Bayes factor (BF) values indicating a decisive support in favor of the presence of a molecular clock for both datasets (i.e., 4 K BF=6405.9; 5' region BF=6244.3). The bModelTest search for the best substitution model to be applied in the Bayesian trees calculation suggested the GTR+G+I one.

The average rate of nucleotide substitution estimated for the central 4 K region of all available PPV isolates, calculated after BEAST elaboration, was 6.91 × 10⁻⁵ subst./site/year (with the 95% highest posterior density (HPD) interval of 4.67 × 10⁻⁵–9.09 × 10⁻⁵). In the Bayesian MCC

tree calculated for the 4 K region (Fig. 3 and Additional Table 4A) the position of the TMRCA was determined at 4546 years ago (95% HPD 3141–6099) when the group of Euro-Med strains separated from the branch leading to PPV-W and the cherry-adapted strains. The separation of PPV-EA from the Euro-Med group could have happened 3655 years ago (95% HPD 2531–4944) while for its part, the separation of the clade of cherry-adapted strains from the W strain is dated back at 3087 years ago (95% HPD 2111–4136). The initial diversification of the Euro-Med group is predicted to have started 1733 years ago (95% HPD 1200–2350). The separation of the M and T strains from PPV-An is suggested to have occurred much more recently, between 289 and 279 years ago, respectively (95% HPD 200–394 and 185–400). Using this dataset, the tentative dating of the separation of the D and Rec strains at 251 years ago (95% HPD 178–338) could also be hypothesized.

Using the 5' fragment encoding the P1 gene (fragment 36–1406 nt), the BEAST elaboration retrieved a subst./s/y rate of 2.55 × 10⁻⁴ for the global alignment of 539 isolates (95% HPD 2.025 × 10⁻⁴–3.084 × 10⁻⁴), which is about 3.6 times faster than that obtained for the 4 K regions and is likely accounted for the lower conservation of the P1. In Fig. 4 (and Additional Table 4B), the root of the tree dated at 1211 years ago (95% HPD 962–1482) and the main diversification of the cherry-adapted strains at 707 years ago (95% HPD 545–872).

Regardless the difference in substitution rates, the Bayesian analysis on both datasets indicated that the separation of the PPV-An strain is anterior to that of the D strain, and thus to all the subsequent node bifurcation events in the Euro-Med group.

Furthermore, the Bayesian elaborations done on both the same regions, but only on a dataset restricted to D isolates (including Rec, for a total of 291 sequences), obtained very similar results, as substitution rates (5' region: 2.029E-4; 4 K region: 8.688E-5) and TMRCA datation (5' region: 1409.404 years before present; 4 K region: 3541.299 years before present), thus indicating that the large predominance of the D isolates in the dataset could substantially have influenced the complete dataset output values.

Parallel to these dating inferences, the two datasets were used in synchronous BEAST elaborations assuming

(See figure on next page.)

**Fig. 3** Time-scaled maximum-clade-credibility tree inferred from the 4 K genome region of 539 selected PPV isolates. Isolates in the tree are collapsed by strain and branch lengths are scaled by distance from TMRCA according to time (years from present), as shown by the reference bar at bottom. At the major nodes, a pie chart shows the host set probability with *Prunus* host species colored according to the legend inset. The pink bar at each node represents the 95% confidence interval (HPD 95%). Specific values for each node date and percentage of host set preference are reported in Additional Table 4A
<figure><img src="image_7.png" /><figcaption>**Fig. 3** (See legend on previous page.)</figcaption></figure>
the same sampling date (i.e. 2017) for all sequences. A total of 800 and 600 millions of iterations were performed on the 4 K and the 5' fragment, respectively, using the same conditions as for the previous elaborations. None of these new tests was able to reach a statistically significant ESS for the Tracer parameters, thus suggesting the meaningfulness of the sampling dates associated to the input sequences and that, even if the initial dataset used for the TRMCA search included portions of full-length recombinant sequences, the temporal signal retrieved was significant.

Interestingly, both MCC trees returned the inferred ancestral host set probability at the nodes of clades bifurcation (Additional Table 4, A and B), since for all the used isolates information on the host plant from which they were characterized was available. The pie charts linked to the main nodes in Figs. 3 and 4 report this information. While most of the nodes of the 5' MCC tree associate the preferential host state with *P. armeniaca* (apricot), the root node of the 4 K MCC tree showed a slightly higher probability signal at 24.3% for the *P. cerasus* (sour cherry) host over *P. armeniaca* (with a probability at 17.9%).

The phylogeographic analysis of the 4 K MCC tree, visualized by SPREAD [55] and based on the currently available dataset of PPV isolates, putatively localized the origin of a PPV ancestor in Russia (Additional Fig. 2). Then, independent introductions in the Mediterranean basin (in Turkey, as the most remote event, and then in Egypt) are postulated. The presence of a wealth of PPV strains (D, M, T and Rec) in Turkey, both in its Asian and European parts [19, 21, 28, 60], suggests Turkey as an important center of differentiation of these strains. The movement of PPV-infected plant material to the Balkans and Central Europe could have then proceeded from Turkey later.

## Discussion

The molecular characterization of the AL-11pl isolate, along with its unique properties, offers further insights into the genetic structure and variability of the plum pox virus strains identified to date [6]. While its genome has a typical potyviral organization that is essentially colinear with that of other PPV isolates, it shows some unusual properties. From the serological point of view, AL11-pl shows reactivity to both

PPV-D-specific (4DG5, [61] and PPV-M-specific (AL, [62]) Mabs. While not unique, such a reactivity pattern is very uncommon and further emphasizes the fact that despite their excellent general effectiveness, PPV strain-specific Mabs cannot be considered as absolutely reliable tools for strain identification [56].

A second unusual feature of AL-11pl is that different phylogenetic affinities with other PPV strains are observed for different parts of its genome. Indeed, its genome is closely related to those of PPV-M isolates from nt position 2686 up to its 3' end, and to those of PPV-T isolates from nt position 1586 up to its 3'end. On the other hand, AL-11pl genome is divergent from all known PPV isolates/strains in its 5' terminal region up to nt 1586. Recombination analysis indicates that this unusual pattern of divergence results from events with breakpoints predicted around nt positions 1561 and 2686.

Glasa and Candresse [17] have described a plurality of recombination events involving the 5' part of PPV genomes in a more restricted dataset than the one used here. They already observed that PPV-M and PPV-D share a homologous 5'genomic region, particularly the segment coding for the P1 gene. While this region is widely recognized as the most divergent among potyviruses [31, 63, 64], it remains highly conserved between PPV-D and the PPV-M and -T strains.

Multiple evolutionary scenarios have been proposed to account for the close relationship of PPV-D, M and T in this 5'genome region, often based on RDP recombination analysis [7, 17, 18]. One of the scenarios put forward by [17], which hypothesized an ancestral strain, having the features similar to PPV-An, as the major parent of PPV-M ad PPV-T, with PPV-D as the minor parent, is the most parsimonious scenario and the one that explains at the best the conservation pattern observed for the P1 gene for the currently known PPV diversity. This does not mean that this scenario necessarily faithfully represents PPV evolutionary history but that it is today our best hypothesis. The possible discovery of further PPV isolates with original properties may necessitate to revisit this scenario in the future or altogether to reject it.

Likewise, some details of the scenario remain to be clarified. In particular, it is not clear whether PPV-M and T are derived from two independent events or whether

(See figure on next page.)

**Fig. 4** Time-scaled maximum-clade-credibility tree inferred from the 5' genomic region (nt positions 36–1406) of 539 selected PPV isolates. Isolates in the tree are collapsed by strain and branch lengths are scaled by distance from TMRCA according to time (years from present), as shown by the reference bar at bottom. At the major nodes, a pie chart shows the host set probability with *Prunus* host species colored according to the legend inset. The pink bar at each node represents the 95% confidence interval (HPD 95%). Specific values for each node date and percentage of host set preference are reported in Additional Table 4B
<figure><img src="image_8.png" /><figcaption>**Fig. 4** (See legend on previous page.)</figcaption></figure>
sequential events were involved (with PPV-T resulting from a secondary recombination between PPV-M and PPV-An or, conversely PPV-M resulting from a secondary recombination between PPV-T and PPV-D, as predicted by our RDP analysis). However, the scenario of two independent events between PPV-An and PPV-D appears as the most parsimonious one, because it involves only two single recombination events as compared to a single and a double recombination for the alternative scenarios.

Algorithms used to detect recombination events do not always give the correct directionality of the detected events, correctly identifying the parents and the derived recombinant(s), but following Ockam's razor preference should be given to the most parsimonious scenario. We therefore consider in what follows PPV-An as the non-recombined ancestor and PPV-M and PPV-T as recombined descendants. We also suspect that the interpretation by the RDP4 analysis of PPV-An as a recombinant could be a consequence of the high number of PPV-M and PPV-T isolates in the dataset, contrasted with the presence of a single PPV-An isolate.

The proper reconstruction of PPV recombination history is important in general terms for our understanding of its evolutionary history. For example, [18] favoured a scenario in which PPV-An and PPV-T are recombinants but PPV-M is not. This led them to use full genomic sequences and a dataset including 28 PPV-M isolates for their analyses and particularly for their estimation of PPV TMRCA. The inclusion of such recombinant isolates has the potential to lead to erroneous conclusions and may be responsible for their consideration that any temporal signal in their PPV dataset is not linearly related to time or to the very old TMRCA (5.5 × 10⁹ years ago with very large 95% CI values).

For our datation analysis, we used all fully sequenced PPV genomes for which metadata were also available. The average substitution rate computed here for the 4 K central genome region, provided a value lower than those reported for several plant and animal viruses and bacteriophages with RNA genomes, that range within an order of magnitude of 10⁻⁴ nucleotide substitutions per site per year [41, 65, 66]. A reason for this difference may be that the analyzed region contains some of the most conserved potyviral genes and may therefore be submitted to stronger selective pressure and evolve slower than the genome as a whole (an extensive analysis of potyviral nucleotide hypervariable regions was done in [64]). Indeed, the substitution rate calculated for the 5' region is higher than that calculated for the 4 K region, conceivably corresponding to the higher variability of the P1 gene. Conversely, the datation of the nodes is more recent when compared to the former region. The shorter sequence length and more recent TMRCA date are in keeping with the finding that a positive correlation was observed between

the shorter dataset sequence length and a more recent age of the TMRCA retrieved [33]. Moreover, the 5' dataset is characterized by the presence of prevailing PPV-D-derived sequences, and we know by the 4 K region MCC dated tree that PPV-D is the most recent non-recombined strain to appear. Therefore, the influence of such D-strain bias in addition to the "faster" substitution rate in determining a most recent TMRCA for the 5' region cannot be ruled out.

The datation reported here using the 4 K region is in agreement with that estimated for PPV by Gibbs et al. [67], since, using only the core portion of the coat protein gene and the 3' non-coding region on a different isolate dataset and finding a slightly higher substitution rate, they estimated a time of radiation of 4532 years ago, which is remarkably close to the value we obtained using the 4 K region and a much larger dataset. Moreover, [42] calculated a date of radiation for the potyvirus genus ranging from 6560 (if the coherently-evolving CP fragment was used) to 7250 years ago (if the complete polyprotein-coding sequence was used). These values are, however, older than the PPV TMRCA calculated by [18] using the different sub-tree height comparison method [68] on the similar PVY background sequences [34].

The estimated divergence times calculated using the 4 K region suggests that PPV-M and PPV-T separated from PPV-An approximately three centuries ago, suggesting that the recombination events leading to the emergence of those two strains may have occurred around the same time. PPV-Rec emerged from a slightly more recent recombination event (around 250 years ago) involving PPV-M and a PPV-D isolates. The emergence of PPV-Rec in the Balkans is particularly well supported. [69] and [29] have indeed suggested that isolates of M-Istanbul subgroup of M strain could be the parents of Rec strain, due to shared mutation in the CP gene.

These timing estimates for the key evolutionary steps in PPV history have been obtained using the largest possible dataset and an internal genome region that does not contain recombination breakpoints according to the most parsimonious evolutionary scenario. They should however still be considered with caution, because of the large extrapolation involved, given that the earliest timed PPV sequences in the dataset only goes back to the 1980's or less than 40 years ago and given there are still many questions about the stability of viral evolutionary rates over extended time periods or following major transition events such as recombination. However, we could obtain a temporal signal evidence for both our datasets when sampling dates were exposed and not through a synchronic simulation or after the artificial randomization of the same sampling dates obtained by the DRT test. In addition, the presence of the molecular clock in the studied dataset was further validated by Bayes factor
calculation on the MLE values obtained through PS/SS analysis.

The phylogeographic diffusion in discrete space analysis (Additional Fig. 2) suggests that the PPV TMRCA could have originated in southern European Russia (i.e. around the Volga basin, or close to the northern borders of the Black Sea, the Caucasus or the Caspian Sea). The model then predicts that PPV spread southwest to Egypt to a limited extent, and to Turkey, which became a secondary center of diversification, giving rise, in Turkey or upon further spread to neighbouring areas such as the Balkans, to other Euro-Mediterranean strains (An and D first and thereafter the recombinant strains M, T and Rec). The Ottoman Empire's expansion from the fourteenth century CE may have facilitated the movement of PPV strains across these regions. In parallel, PPV isolates in north-eastern Russian regions could have further differentiated, leading to the W strain and, in a major host adaptive jump, to all the cherry-adapted strains [15, 70]. Indeed, PPV-C, -CV and -CR strains, together with other cherry-adapted phylogroups appear widespread in the southern part of the Volga Basin and Tatarstan [15], while PPV-W was also found in Khazakstan [71] and in several parts of Russia.

Alternative phylogeographic scenarios remain possible, in particular those placing the origin of PPV in the Balkans or in Turkey, two areas with very high PPV diversity. Since such phylogeographic analyses are strongly influenced by the available dataset, future discoveries could very well reshape the spread hypothesis. This analysis also suggests that the recombination events leading to PPV-M and PPV-T are significantly anterior to the first report of Sharka disease symptoms in *Prunus* in Bulgaria almost 100 years ago [72].

An analysis of host range evolution in potyviruses [73] pointed out that the host association preferentially derived through jumps due to the local insect transmission more than co-divergence within ancestral hosts. Notably these authors reported the existence of close phylogenetic affinities between PPV and two monocot-infecting potyviruses, hyacinth mosaic virus and asparagus virus 1 and [18] and [42] have also suggested that PPV may have derived from a wild monocot-infecting ancestor. Here, the elaboration of the ancestral host set probability from the *Prunus* species present in our metadata gave always the highest likelihood to apricot, except for the root node in the 4 K MCC tree in which sour cherry had a slightly higher probability before apricot (24.3% vs 17.9%, respectively). However, such a result is very highly influenced by the composition and structure the dataset used and may be dramatically reshaped by discovery of a single original isolate, as exemplified by the rethinking of turnip mosaic virus evolutionary

history caused by the discovery of hedge mustard mosaic virus [74]. In this respect, it should be kept in mind that a scenario proposing cherry as the ancestral host of PPV would in fact be less parsimonious than the one envisioning a prunus non-cherry host and a single host jump leading to all cherry adapted strains.

Despite sharing about 80% of their genomes, PPV-An and PPV-M/-T have had very different evolutionary success. In the largest coordinated PPV survey in Europe (SharCo project 2006–2011), only one isolate (AL-11pl) was identified as PPV-An among ~800 sampled PPV isolates from multiple countries, suggesting that PPV-M and -T hold significant evolutionary advantages/fitness over their non-recombined ancestor. Notably, the three most successful and widespread PPV strains (D, M, and Rec) share the same 1–1561 genomic region, including the P1 and part of the HC-Pro genes. This suggests that one or both genes may have contributed to their success, possibly by enhancing adaptation to *Prunus* hosts. In this respect it could be noteworthy that PPV P1 has been shown to influence infection and symptoms expression in some herbaceous hosts [63].

## Conclusions

In the present study, we examined the phylogeny and recombination history of the AL11-pl isolate, which typifies the PPV-An strain, in the context of the entire background of PPV isolates so far available. The correct evaluation of PPV diversification and recombination history enabled a more comprehensive phylogenetic analysis of the PPV strains, which led us to propose that PPV-An, along with PPV-D, is the previously unidentified progenitor of the prevalent Euro-Mediterranean recombinant strains (e.g., M, Rec, and T). The Bayesian inference of dating in a recombination-neutral region placed the PPV TMRCA in line with the described history of potyviruses radiation at the origin of agricultural practices. Taken together the results presented here highlight PPV-An as a key player in PPV evolutionary history and consolidate PPV as a promising model to study host-adaptive evolution processes and phylogeography among the most damaging viruses for agricultural systems.

## Abbreviations

<table><tr><td>BEAST</td><td>Bayesian evolutionary analysis by sampling trees</td></tr><tr><td>BF</td><td>Bayes factor</td></tr><tr><td>BSSVS</td><td>Bayesian stochastic search variable selection</td></tr><tr><td>CI</td><td>Credible interval</td></tr><tr><td>DRT</td><td>Date-randomization test</td></tr><tr><td>ESS</td><td>Effective sampling size</td></tr><tr><td>HC-Pro</td><td>Helper component proteinase</td></tr><tr><td>HPD</td><td>Highest posterior density</td></tr><tr><td>iTOL</td><td>Integrative tree of live</td></tr><tr><td>kDa</td><td>Kilo dalton</td></tr><tr><td>Mab</td><td>Monoclonal antibody</td></tr><tr><td>MAFFT</td><td>Multiple alignment using fast fourier transform</td></tr><tr><td>MCC</td><td>Maximum clade credibility</td></tr></table>
<table><tr><td>MCMC</td><td>Markov Chain Monte Carlo</td></tr><tr><td>MEGA</td><td>Molecular Evolutionary Genetics Analysis software</td></tr><tr><td>ML</td><td>Maximum likelihood</td></tr><tr><td>MLE</td><td>Marginal likelihood estimation</td></tr><tr><td>NCR</td><td>Non-coding region</td></tr><tr><td>Nla-Pro</td><td>Nuclear inclusion a-protease</td></tr><tr><td>ORF</td><td>Open reading frame</td></tr><tr><td>PIPO</td><td>Pretty interesting potyvirus ORF</td></tr><tr><td>PPV</td><td>Plum pox virus</td></tr><tr><td>PPV-An</td><td>PPV-Ancestor</td></tr><tr><td>PPV-C</td><td>PPV-Cherry</td></tr><tr><td>PPV-CR</td><td>PPV-Cherry Russia</td></tr><tr><td>PPV-CV</td><td>PPV-Cherry Volga</td></tr><tr><td>PPV-D</td><td>PPV-Dideron</td></tr><tr><td>PPV-EA</td><td>PPV-El Amar</td></tr><tr><td>PPV-M</td><td>PPV-Marcus</td></tr><tr><td>PPV-Rec</td><td>PPV-Recombinant</td></tr><tr><td>PPV-T</td><td>PPV-Turkey</td></tr><tr><td>PPV-W</td><td>PPV-Winona</td></tr><tr><td>PS/SS</td><td>Path sampling/stepping-stone sampling</td></tr><tr><td>PVY</td><td>Potato virus Y</td></tr><tr><td>RDP</td><td>Recombination detection program</td></tr><tr><td>RNA</td><td>Ribonucleic acid</td></tr><tr><td>TMRCA</td><td>The most recent common ancestor</td></tr><tr><td>TuMV</td><td>Turnip mosaic virus</td></tr><tr><td>VPg</td><td>Viral protein genome linked</td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12985-025-02892-7.

Additional file 1: Fig. 1. Maximum likelihood phylogeny of 539 PPV full length genomes. Strains or phylogroups are indicated as follows: PPV-T, PPV-An, PPV-M, PPV-EA, PPV-W, PPV-C, PPV-Y, PPV-CV, PPV-SC, PPV, PPV-CR, PPV-Dand PPV-Rec. Colored spots correspond to the *Prunus* species from which isolates were identified, as indicated in the legend. The bootstrap values at the main nodes are not reported in the pictures to improve visualization, since most of them are > = 60.

Additional file 2: Fig. 2. History and main routes of the PPV dispersal as predicted by SPREAD program. The map displays the virus movement from the putative center of origin (Southern European Russia, 2523 before common era: 4546 years from present) to Mediterranean countries (Turkey, 397 common era: 1626 years from present; Egypt, 1784 common era: 239 years from present). The arrows indicate the direction of the movement. The red star marks the putative center of origin, the yellow star a further region of diversification, while white stars (the Balkans and Central Europe, and then the Far East) the settlement and the current presence of the virus as a consequence of trade of infected planting materials. Further and recent routes of westward incursions (i.e. to the Americas) are not shown.

Additional file 3: Fig. 3. Summary of the date-randomization tests of the data set used for tip-dating analysis in this study. A) PPV genome region: 36–1400, and B) PPV genome region: 3500–7500. Estimates of nucleotides substitution rate (substitution/site/year) from the original data set and ten permutations. Symbols represent the mean rate estimate for each data set, with error bars showing the 95% credible intervals (CI). Symbols and error bars in red color represent the estimate from the original data set, whereas those in blue color represent estimates from date-randomized data sets. The 95% CI of the estimates from the data-randomization replicates did not overlap with the mean posterior estimate from the original dataset, indicating the presence of temporal signal.

Additional file 4: Table 1. List of the 539 full-genome PPV isolates used in the present study. These isolates were selected among the 609 isolates with complete genomes retrieved from GenBank in November 2023 because complete metadata information (country and host of origin, sampling year) was available for them. Format and internal code (PPnnn) were imposed for subsequent software operations. Host, country and strain are indicated for each isolate.

Additional file 5: Table 2. Main recombination events identified by the RDP4 analysis for the dataset of 539 full-genome PPV isolates. For events impacting several recombinant isolates only an exemplar isolate is reported. Probability significance values (p-values) are reported according to the specific detection methods. Table key as from the RDP4 output.

Additional file 6: Table 3. Output from TempTest Software using full-lenght PPV genomes.

Additional file 7: Table 4. Output of datation, statistical values and host preference obtained from the Bayesian inference analysis. Age of predicted nodes bifurcation is indicated as years from present. 4A) Values from elaboration of the MCC tree for the 4 K genomic region. The mean substitution rate per site per year and the related 95% HPD interval are highlighted. Pie charts of the host set probability for the main nodes with the specific percentage values are also represented in Fig. 3. 4B) Values from elaboration of the MCC tree for the 5' genomic region (nt 36–1406). The mean substitution rate per site per year and the related 95% HPD interval are highlighted. Pie charts of the host set probability for the main nodes with the specific percentage values are also represented in Fig. 4.

## Acknowledgements

We thank SHARCO project (Containment of Sharka virus in view of EU-expansion, 2006-2011) funded under specific Programme FP7"Cooperation": Food, Agriculture and Biotechnology (grant agreement ID: 204429) for supporting field surveys performed in Albania, and which allowed the identification of several PPV strains, including PPV-An. In particular, the authors wish to thank Drs. Donato Boscia and Arben Myrta which collected these isolates. A kind acknowledgment is also due to Dr. Saverio Vicario for the critical reading of the manuscript and helpful suggestions. The authors would like to remember the passionate commitment and dedication to the study and containment of Plum pox virus operated by the passed Professor Vito Nicola Savino (University of Bari), who was a constant and helpful reference for the researchers in this field. We thank Japan Society for the Promotion of Science Exchange Research Program for supporting S.K. to visit V. P. lab. We thank the project PNRR "OnFood" and the project SaveGrainPugliaLeg for contribution to facilities used. We are grateful to Prof. Chikara Masuta, Faculty of Agriculture, University of Hokkaido (Japan), for his support and critical reading.

## Authors' contributions

S.K. prepared and interpreted the sequence dataset; F.P. sequenced the An isolate and did the preliminary sequence analysis and comparison; T.C. and A.M. conceptualized the research; M.C., V.P., T.C. and A.M. wrote the manuscript; PL and V.P. took care of data repository and prepared pictures and tables; MC did the Bayesian elaborations on the dataset. All authors read, edited and approved the final manuscript.

## Funding

Japan Society for the Promotion of Science, JP22J22297, FP7 Cooperation, 204429, 204429.

## Data availability

Data is provided within the manuscript or supplementary information files.

## Declarations

### Ethics approval and consent to participate

Not applicable.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

### Author details

<footnote n="1">¹Department of Biology Agricultural and Food Sciences, Institute for Sustainable Plant Protection, National Research Council, Bari Unit, 70126 Bari, Italy.</footnote>

<footnote n="2">²Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589,</footnote>
Japan. ³UMR 1332 Biologie du Fruit Et Pathologie, INRAE - Université de Bordeaux, CS20032, 33882 cedex Villenave d'Ornon, France. ⁴Present Address: Regional Phytosanitary Service - Apulia Region, Lungomare Nazario Sauro, 51, Bari 70100, Italy. ⁵Present Address: Division of Systems Virology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Received: 18 May 2025 Accepted: 23 July 2025

Published online: 01 October 2025

## References

1. Revers F, García JA. Molecular biology of potyviruses. Adv Virus Res. 2015;92:101–99.

2. James D, Thompson D. Hosts and symptoms of Plum pox virus: ornamental and wild Prunus species. EPPO Bull. 2006;36:222–4.

3. Sochor J, Babula P, Adam V, Krska B, Kizek R. Sharka: the past, the present and the future. Viruses. 2012;4:2853–901.

4. García JA, Cambra M. Plum pox virus and sharka disease. Plant Viruses. 2007;1:69–79.

5. Cambra M, Madariaga M, Varveri C, Çağlayan K, Morca Af, Chirkov S, et al. Estimated costs of plum pox virus and management of sharka, the disease it causes. Phytopathol Mediterr. 2024. https://doi.org/10.3625/phyto-15581.

6. García JA, Rodamilans B, Martínez-Turiño S, Valli AA, Simón-Mateo C, Cambra M. Plum pox virus: An overview of the potyvirus behind sharka, a harmful stone fruit disease. Ann Appl Biol. 2025;186:49–75.

7. García JA, Glasa M, Cambra M, Candresse T. *P. lum pox virus* and sharka: a model potyvirus and a major disease. Mol Plant Pathol. 2014;15:226–41.

8. Scholthof K-BG, Adkins S, Czosnek H, Palukaitis P, Jacquot E, Hohn T, et al. Top 10 plant viruses in molecular plant pathology. Mol Plant Pathol. 2011;12:938–54.

9. Labonne G, Yvon M, Quiot JB, Avinent L, Llacer G. Aphids as potential vectors of plum pox virus: comparison of methods of testing and epidemiological consequences. In: XVI International Symposium on Fruit Tree Virus Dis. 1994;386:207–18. Available from: https://www.actahort.org/books/386/386_27.htm. Cited 2025 Mar 3.

10. Wetzel T, Candresse T, Ravelonandro M, Delbos RP, Mazyad H, Aboul-Ata AE, et al. Nucleotide sequence of the 3'-terminal region of the RNA of the El Amar strain of plum pox potyvirus. J Gen Virol. 1991;72:1741–6.

11. Nemchinov L, Hadidi A, Maiss E, Cambra M, Candresse T, Damsteegt V. Sour cherry strain of plum pox potyvirus (PPV): molecular and serological evidence for a new subgroup of PPV strains. Phytopathology. 1996;86:1215–21.

12. James D, Varga A. Nucleotide sequence analysis of Plum pox virus isolate W3174: evidence of a new strain. Virus Res. 2005;110:143–50.

13. Serçe ÇU, Candresse T, Svanella-Dumas L, Krizbai L, Gazel M, Çağlayan K. Further characterization of a new recombinant group of Plum pox virus isolates, PPV-T, found in orchards in the Ankara province of Turkey. Virus Dis. 2009;142:121–6.

14. Chirkov S, Ivanov P, Sheveleva A, Zakubanskiy A, Osipov G. New highly divergent Plum pox virus isolates infecting sour cherry in Russia. Virology. 2017;502:56–62.

15. Chirkov S, Sheveleva A, Gasanova T, Kwon D, Sharko F, Osipov G. New cherry-adapted plum pox virus phylogroups discovered in Russia. Plant Dis. 2022;106:2591–600.

16. Chirkov S, Ivanov P, Sheveleva A. Detection and partial molecular characterization of atypical plum pox virus isolates from naturally infected sour cherry. Arch Virol. 2013;158:1383–7.

17. Glasa M, Candresse T. Partial sequence analysis of an atypical Turkish isolate provides further information on the evolutionary history of Plum pox virus (PPV). Virus Res. 2005;108:199–206.

18. Hajizadeh M, Gibbs AJ, Amirnia F, Glasa M. The global phylogeny of plum pox virus is emerging. J Gen Virol. 2019;100:1457–68.

19. Teber S, Ceylan A, Gürcan K, Candresse T, Ulubaş Serçe Ç, Akbulut M. et al. Genetic diversity and molecular epidemiology of the T strain of *Plum pox virus*. Plant Pathol. 2019;68:755–63.

20. Teber S, Gürcan K, Brevet M, Palmisano F, Candresse T, Dallot S. High genetic diversity suggests a long evolutionary history of plum pox virus-M in Turkey. Plant Pathol. 2023;72:300–11.

21. Gürcan K, Teber S, Candresse T. Genetic analysis suggests a long and largely isolated evolutionary history of plum pox virus strain D in Turkey. Plant Pathol. 2020;69:370–8.

22. Kerlan C, Dunez J. Differenciation biologique et serologique de souches du virus de la Sharka. Annales de Phytopathologie. 1979. Available from: https://agris.fao.org/search/en/providers/123819/reCORDS/64735fe32c1d629bc97e00d0. Cited 2025 Feb 27.

23. Myrta A, Varga A, James D. The complete genome sequence of an El Amar isolate of plum pox virus (PPV) and its phylogenetic relationship to other PPV strains. Arch Virol. 2006;151:1189–98.

24. Palmisano F, Boscia D, Minafra A, Myrta A, Candresse T. An atypical Albanian isolate of Plum pox virus could be the progenitor of the Marcus strain. 2012. Available from: https://agris.fao.org/search/en/providers/122439/records/647471d1bf943c8c798213f0. Cited 2025 Feb 27.

25. Glasa M, Prikhodko Y, Predajňa L, Nagyová A, Shneyder Y, Zhivaeva T, et al. Characterization of sour cherry isolates of *Plum pox virus* from the Volga Basin in Russia reveals a new cherry strain of the virus. Phytopathology. 2013;103:972–9.

26. Sihelská N, Glasa M, Šubr ZW. Host preference of the major strains of plum pox virus—opinions based on regional and world-wide sequence data. J Integr Agric. 2017;16:510–5.

27. Maejima K, Hashimoto M, Hagiwara-Komoda Y, Miyazaki A, Nishikawa M, Tokuda R, et al. Intra-strain biological and epidemiological characterization of plum pox virus. Mol Plant Pathol. 2020;21:475–88.

28. Gürcan K, Teber S, Çağlayan K. Further investigation of a genetically divergent group of plum pox virus-M strain in Turkey. J Plant Pathol. 2019;101:385–91.

29. Glasa M, Marie-Jeanne V, Labonne G, Šubr Z, Kúdela O, Quiot J-B. A natural population of recombinant plum pox virus is viable and competitive under field conditions. Eur J Plant Pathol. 2002;108:843–53.

30. Glasa M, Malinowski T, Predajňa L, Pupola N, Dekena D, Michalczuk L, et al. Sequence variability, recombination analysis, and specific detection of the W strain of *Plum pox virus*. Phytopathology. 2011;101:980–5.

31. Maliogka V, Salvador B, Carbonell A, Sáenz P, San León D, Oliveros JC, et al. Supplementary information for Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features. Mol Plant Pathol. 2012;13(8):877–86.

32. Palmisano F, Minafra A, Myrta A, Boscia D. First report of Plum pox virus strain PPV-T in Albania. J Plant Pathol. 2015;97. https://search.ebscohost.com/login.aspx?direct=true&profile=ehost&scope=site&authtype=crawler&jrn1=11254653&AN=113631464&h=Ud%2BtVtM9RelbpxnYL3KiuOe6N8weqZ1T2f%2FTYH9AOWvhc9sCFpAGOz8vRxO9hJ%62FXfBpb70j0b43Kw9X

33. Gibbs AJ, Hajizadeh M, Ohshima K, Jones RA. The potyviruses: an evolutionary synthesis is emerging. Viruses. 2020;12:132.

34. Gao F, Kawakubo S, Ho SY, Ohshima K. The evolutionary history and global spatio-temporal dynamics of potato virus y. Virus Evol. 2020;6:veaa056.

35. Yasaka R, Fukagawa H, Ikematsu M, Soda H, Korkmaz S, Golnaraghi A, et al. The timescale of emergence and spread of turnip mosaic potyvirus. Sci Rep. 2017;7:4240.

36. Kawakubo S, Gao F, Li S, Tan Z, Huang Y-K, Adkar-Purushothama CR, et al. Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road. Proc Natl Acad Sci USA. 2021;118: e2021221118.

37. Probowati W, Kawakubo S, Ohshima K. Narcissus plants: a melting pot of potyviruses. Viruses. 2022;14:582.

38. Kawakubo S, Kim H, Takeshita M, Masuta C. Host-specific adaptation drove the coevolution of leek yellow stripe virus and *Allium* plants. Burbank LP, (ed). Microbiol Spectr. 2023;11:e02340–23.

39. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012;29:1969–73.

40. Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol. 2007;7:214.

41. Duffy S, Shackelton LA, Holmes EC. Rates of evolutionary change in viruses: patterns and determinants. Nat Rev Genet. 2008;9:267–76.

42. Gibbs AJ, Fargette D, Garcia-Arenal F, Gibbs MJ. Time - the emerging dimension of plant virus studies. J Gen Virol. 2010;91:13–22.
43. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.

44. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9.

45. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-tree 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37:1530–4.

46. Letunic I, Bork P. Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–6.

47. Martin DP, Murrell B, Golden M, Khoosal A, Muhire B. RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evol. 2015;1:vew003.

48. Rambaut A, Lam TT, Max Carvalho L, Pybus OG. Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). Virus Evol. 2016;2: vew007.

49. Sagulenko P, Puller V, Neher RA. TreeTime: Maximum-likelihood phylogenetic analysis. Virus Evol. 2018;4: vex042.

50. Bouckaert RR, Drummond AJ. bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evol Biol. 2017;17:42.

51. Baele G, Lemey P, Bedford T, Rambaut A, Suchard MA, Alekseyenko AV. Improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty. Mol Biol Evol. 2012;29:2157–67.

52. Suchard MA, Lemey P, Baele G, Ayres DL, Drummond AJ, Rambaut A. Bayesian phylogenetic and phylogen dynamic data integration using BEAST 1.10. Virus Evol. 2018;4: vey016.

53. Wang L-G, Lam TT-Y, Xu S, Dai Z, Zhou L, Feng T, et al. Treeio: an R package for phylogenetic tree input and output with richly annotated and associated data. Mol Biol Evol. 2020;37:599–603.

54. Yu G, Smith DK, Zhu H, Guan Y, Lam TT. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. McInerny G, (ed). Methods Ecol Evol. 2017;8:28–36.

55. Bielejec F, Rambaut A, Suchard MA, Lemey P. SPREAD: spatial phylogenetic reconstruction of evolutionary dynamics. Bioinformatics. 2011;27:2910–2.

56. Candresse T, Cambra M, Dallot S, Lanneau M, Asensio M, Gorris MT, et al. Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the D and M serotypes of plum pox potyvirus. Phytopathology®, 1998;88:198–204.

57. Chung BY-W, Miller WA, Atkins JF, Firth AE. An overlapping essential gene in the Potyviridae. Proc Natl Acad Sci USA. 2008;105:5897–902.

58. Urcuqui-Inchima S, Haenni A-L, Bernardi F. Potyvirus proteins: a wealth of functions. Virus Res. 2001;74:157–75.

59. Wang Y, Gal-On A, Huet H, Kadoury D, Raccah B, Peng YH. Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. J Gen Virol. 1998;79:897–904.

60. Gürcan K, Teber S, Akbulut M, Çağlayan K. Genetic diversity and a long evolutionary history of plum pox virus strain rec in Turkey. Eur J Plant Pathol. 2021;161:453–61.

61. Cambra M, Asensio M, Gorris MT, Pérez E, Camarasa E, Garciá JA, et al. Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins¹. EPPO Bull. 1994;24:569–77.

62. Boscia D, Zeramdini H, Cambra M, Potere O, Gorris MT, Myrta A, et al. Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus. Eur J Plant Pathol. 1997;103:477–80.

63. Shan H, Pasin F, Valli A, Castillo C, Rajulu C, Carbonell A, et al. The Potyviridae P1a leader protease contributes to host range specificity. Virology. 2015;476:264–70.

64. Nigam D, LaTourrette K, Souza PF, Garcia-Ruiz H. Genome-wide variation in potyviruses. Front Plant Sci. 2019;10:1439.

65. Sanjuán R, Agudelo-Romero P, Elena SF. Upper-limit mutation rate estimation for a plant RNA virus. Biol Lett. 2009;5:394–6.

66. Jenkins GM, Rambaut A, Pybus OG, Holmes EC. Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis. J Mol Evol. 2002;54:156–65.

67. Gibbs AJ, Ohshima K, Phillips MJ, Gibbs MJ. The prehistory of potyviruses: their initial radiation was during the dawn of agriculture. PLoS ONE. 2008;3:e2523.

68. Mohammadi M, Hosseini A, Nasrollanejad S. In silico identification of two novel viruses on Iranian pistachio. Iran J Plant Pathol. 2021;57:81–5.

69. Glasa M, Palkovics L, Komínek P, Labonne G, Pittnerová S, Kúdela O, et al. Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup. J Gen Virol. 2004;85:2671–81.

70. Sheveleva A, Glasa M, Kudryavtseva A, Ivanov P, Chirkov S. Genetic diversity, host range and transmissibility of CR isolates of Plum pox virus. J Gen Plant Pathol. 2019;85:39–43.

71. Dallot S, Karychev R, Dolgikh S, Thébaud G, Jacquot E, Decroocq V. First report of Plum pox virus strain W in Kazakhstan, on *Prunus domestica*. Plant Dis. 2019;103:2702.

72. Atanasoff D. Mosaic of stone fruits. 1935. Available from: https://www.cabidigitallibrary.org/doi/full/10.5555/19350501302. Cited 2025 Feb 27.

73. Moury B, Desbiez C. Host range evolution of potyviruses: a global phylogenetic analysis. Viruses. 2020;12:111.

74. Tsarmpopoulos I, Marais A, Faure C, Theil S, Candresse T. A new potyvirus from hedge mustard (*Sisymbrium officinale* (L.) Scop.) sheds light on the evolutionary history of turnip mosaic virus. Arch Virol. 2023;168: 14.

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