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The promiscuous evolutionary history of the family Bromoviridae

Francisco M. Codoñer

Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain

Correspondence
Santiago F. Elena
sfelena{at}ibmcp.upv.es

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Recombination and segment reassortment are important contributors to the standing genetic variation of RNA viruses and are often involved in the genesis of new, emerging viruses. This study explored the role played by these two processes in the evolutionary radiation of the plant virus family Bromoviridae. The evolutionary history of this family has been explored previously using standard molecular phylogenetic methods, but incongruences have been found among the trees inferred from different gene sequences. This would not be surprising if RNA exchange was a common event, as it is well known that recombination and reassortment of genomes are poorly described by standard phylogenetic methods. In an attempt to reconcile these discrepancies, this study first explored the extent of segment reassortment and found that it was common at the origin of the bromoviruses and cucumoviruses and at least at the origin of alfalfa mosaic virus, American plum line pattern virus and citrus leaf rugose virus. Secondly, recombination analyses were performed on each of the three genomic RNAs and it was found that recombination was very common in members of the genera Bromovirus, Cucumovirus and Ilarvirus. Several cases of recombination involving species from different genera were also identified. Finally, a phylogenetic network was constructed reflecting these genetic exchanges. The network confirmed the taxonomic status of the different genera within the family, despite the phylogenetic noise introduced by genetic exchange.

Present address: Evolutionary Genetics and Bioinformatics Laboratory, Department of Genetics, Smurfit Institute of Genetics, University of Dublin, Trinity College, Dublin, Ireland.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Recombination and segment reassortment are important mechanisms for the genesis of new genetic variability in RNA virus populations. Proportionally, the amount of genetic variability produced after a single recombination event is larger than that produced by single point mutations. Although the importance of recombination was underappreciated in early studies of virus genome evolution, it is now recognized as a widespread phenomenon among positive-strand RNA viruses in animals (Grassly & Holmes, 1997; Holmes et al., 1999; Wilson et al., 1988) and plants (Nagy & Bujarski, 1993; Revers et al., 1996; Aranda et al., 1997; Olsthoorn et al., 2002; Bousalem et al., 2003; Cheng & Nagy, 2003; Moreno et al., 2004; Tan et al., 2004; Bonnet et al., 2005; Urbanowicz et al., 2005; Chare & Holmes, 2006; Weng et al., 2007), as well as in retroviruses such as human immunodeficiency virus type 1 (Worobey & Holmes, 1999; Prljic et al., 2004; Althaus & Bonhoeffer, 2005; Galetto & Negroni, 2005), although it is a rare or even non-existent phenomenon among negative-strand viruses (Chare et al., 2003). There are several mechanisms by which RNA recombination may take place, the most common of which is homologous recombination (Lai, 1992). During this process, the donor replaces a highly homologous region in the acceptor, with the recombinant retaining exactly the same genomic organization of the parental RNA molecules. Therefore, the term homologous refers not only to the presence of sequence homology between both parental RNAs but also the necessity of homologous and comparable sites in both parental molecules for the crossovers to take place (Lai, 1992). Aberrant homologous and non-homologous recombinations are less frequent in nature (Lai, 1992).

Amongst positive-strand plant RNA viruses, homologous recombination has been reported in vivo and in vitro during mixed infections (Bousalem et al., 2003; Cheng & Nagy, 2003; Moreno et al., 2004; Tan et al., 2004; Bonnet et al., 2005; Urbanowicz et al., 2005; Weng et al., 2007). Focusing on the family Bromoviridae, reports include recombination events in members of the genera Cucumovirus (Férnandez-Cuartero et al., 1994; Aranda et al., 1997; Fraile et al., 1997; Roossinck et al., 1999; Chen et al., 2002; Bonnet et al., 2005) and Bromovirus (Bujarski & Kaesberg 1986; Allison et al., 1990), as well as between viruses belonging to different genera (de Wispelaere et al., 2005).

In addition to these recombination events, reassortment of genomic RNAs during mixed infection events of multipartite viruses is of extreme importance in the genesis of new strains. In certain cases, these new strains may show completely different virological properties, recent examples of which have been seen in H5N1 influenza A virus (Macken et al., 2006) and the begomovirus species complex responsible for the cassava mosaic disease (Lefeuvre et al., 2007). Segment reassortment has been observed among cucumoviruses (White et al., 1995), although the frequency of such heterologous segregations may be low (Fraile et al., 1997).

The Bromoviridae is one of the most important families of plant RNA viruses, with members distributed worldwide. It has a wide host range (more than 10 000 species) and is the cause of agronomically important diseases. The genome of members of the family Bromoviridae is composed of three segments of positive-sense, single-stranded RNA of approximately 8 kb. RNAs 1 and 2 are monocistronic and encode the proteins involved in virus replication (P1 and P2), whereas RNA 3 encodes the movement (MP) and coat (CP) proteins, the latter of which is translated from the subgenomic RNA 4. Some members of the family (especially cucumoviruses and some ilarviruses) encode a fifth protein (P2b) that is located in RNA 2 and is part of the C-terminal region of the P2 protein. This peptide is involved in silencing suppression, systemic movement and expression of symptoms (Ji & Ding, 2001; Shi et al., 2002).

In recent years, doubts have been cast on the biological significance of the currently accepted official taxonomy of the family Bromoviridae (Rampitsch & Eastwell, 1997; Sánchez-Navarro & Pallás, 1997; Scott et al., 1998; Shiel & Berger, 2000; Codoñer et al., 2005; Codoñer & Elena, 2006). To provide more clues for a better understanding of the taxonomy of this family, we undertook a systemic analysis of the potential roles of recombination and segment reassortment during mixed infections.

	METHODS

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RNA and protein sequences.
Table 1 contains a list of virus species belonging to the family Bromoviridae that were used for this study. Sequences were obtained from the NCBI database (www.ncbi.nlm.nih.gov). Subgroups within the genus Ilarvirus were defined as in Codoñer & Elena (2006).

Phylogenetic reconstruction.
A molecular evolutionary model was first fitted to each of the protein and RNA alignments using PROTTEST v1.0.6 (Abascal et al., 2005) and MODELTEST (Posada & Crandall, 1998), respectively. For the MODELTEST analysis, PAUP v4.0b10 (Swofford, 1998) was used.

Phylogenetic trees for proteins were obtained using the PHYML program implemented in PROTTEST that builds the best tree under the best molecular evolutionary model. Phylogenetic trees for RNA sequences inferred by the best model of nucleotide substitutions were obtained using the CODEML program from PAML v4.0 (Yang, 2007). We accepted the best tree as the one explaining the evolution of each RNA segment with the lowest log-likelihood value. CODEML was also used to obtain the log-likelihood value for the different trees.

RNA segment reassortment analyses.
To look for RNA segment reassortment during mixed infections, a phylogenetic congruence analysis was performed. If segments have co-speciated, i.e. always segregated together, then the tree topologies obtained from different segments must be congruent. In contrast, if reassortment events have taken place during the evolution of the family, the tree topologies will show incongruence among segments. Non-random associations between all three possible pairs of RNAs were determined by reconciling the corresponding phylogenies using a heuristic search in TREEMAP v1.0a (Page, 1994). This approach minimizes the number of independent segregation events among virus segments. The statistical significance of the phylogenetic associations was assessed by performing 10 000 randomizations of both trees using the proportional-to-distinguishable method (Page, 1994). Four different processes affect the relationship between segments (Page, 1994): (i) a co-speciation event takes place when parallel cladogenesis occurs for all segments, i.e. the split of an ancestral species into two new ones maintains the linkage between all three segments; (ii) a duplication event occurs when cladogenesis does not affect all segments at the same time, i.e. after the split of an ancestral species into two new ones, one of the segments speciates but the other remains common among the two new species; (iii) a switch event takes place when a segment from one virus is acquired by a different one; a priori, this should be neglected as no case of gaining an extra segment has been reported; (iv) a sorting event has happened when segments differentially sort during a co-infection process; this type of event is the key support for the reassortment hypothesis.

Recombination analyses.
In addition to reassortment events, recombination within a segment can also seriously influence the branching order and branch lengths of phylogenetic trees (Posada & Crandall, 2002). The presence of recombination events within each RNA segment was explored using RDP v3.22β (Martin et al., 2005b). RDP incorporates several published recombination detection methods into a single suite of tools. Once all potential recombination events are identified, RDP sorts the results and determines the number of unique recombination events identifiable, determining the daughter and parental sequences and the break points. The programs used in this study were: RDP (Martin & Rybicki, 2000), GENECONV (Padidam et al., 1999), BOOTSCAN (Martin et al., 2005a), MAXCHI (Smith, 1992), CHIMAERA (Posada & Crandall, 2001), SISCAN (Gibbs et al. 2000), 3SEQ (Boni et al., 2007) and LARD (Holmes et al., 1999). In all cases, default parameters were used. Only events predicted by half of the methods were considered as significant; this ensured that our approach remained conservative.

The likelihood of predicted recombination events is discussed with regard to the possibility of co-infecting the same host plant by the parental viruses. A list of susceptible hosts for each virus can be obtained from the Plant Virus Online database (http://image.fs.uidaho.edu/vide/refs.htm).

Phylogenetic networks.
Because reassortment and recombination produce networks of sequences rather than strictly bifurcating evolutionary trees, as a final step in our analysis a phylogenetic network (Huson & Kloepper, 2005) was constructed from the concatenated alignment of the three genomic RNA segments. This network, obtained by the split decomposition method implemented in SPLITSTREE v4.8 (Huson & Bryant, 2006), depicts parallel edges between sequences when there are conflicting phylogenetic signals.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Fig. 1 shows the phylogenetic trees inferred for the three segments by concatenating the corresponding coding and non-coding regions. A single phylogenetic tree per RNA segment was inferred using CODEML and fitting of heterogeneous substitutions models was carried out as described in Codoñer et al. (2005). The three topologies were not fully congruent. Within the genus Ilarvirus, the association of AMV with PDV (see Table 1 for virus abbreviations) depended on which RNA was analysed: according to RNAs 1 and 3, these two viruses share a common phylogenetic origin whereas, according to RNA 2, PDV is a closer relative of PNRSV and ApMV. The location of CiLRV inferred from RNA 3 was also incongruent with that inferred from the other two RNAs. The location of APLPV was also incongruent: it appeared within the genus Ilarvirus according to RNAs 1 and 3 but clustered outside the genus according to RNA 2. Similarly, the order of clustering within the genus Cucumovirus depended entirely on which RNA was analysed. Finally, the clustering obtained for the three bromoviruses was congruent for RNAs 2 and 3, placing CCMV and BBMV as closer relatives, but clustering with RNA 1 was incongruent.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Phylogenetic trees obtained for the three genomic segments by concatenating coding and non-coding regions and fitting a heterogeneous nucleotide-substitution model using CODEML. See Table 1 for virus abbreviations.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Reconciliated trees for each pair of genomic RNAs (left) and plots of the lengths for the branches where co-speciation events took place (right). (a) RNA 1 (red) and RNA 2 (black): 12 co-speciation (), five duplication (, blue) and 17 reassortment (x) events. (b) RNA 1 (red) and RNA 3 (black): 11 co-speciation, six duplication and 20 reassortment events. (c) RNA 2 (red) and RNA 3 (black): 12 co-speciation, five duplication and 17 reassortment events.

Finally, Fig. 2(c) shows the best reconciled tree for RNAs 2 and 3. Twelve co-speciation events were predicted in this case and the positive correlation between the rates of genetic divergence in the two RNAs suggested concerted evolution in these particular branches (Fig. 2c: r²=0.6439, P<0.0001). Nonetheless, five duplication and 17 reassortment events were required to reconcile both phylogenetic trees (resampling test: P<0.0001).

Pooling together the information from these three analyses, the following picture emerges, showing the importance of reassortment events on the genesis of new viral species. (i) The origin of bromoviruses and cucumoviruses is quite promiscuous, with many such reassortment events. For example, BMV RNA 1 was generated by duplication of an ancestral segment and did not follow the cladogenetic pattern of the other two segments. To explain the evolution of the cucumoviruses, an ancestral duplication and three reassortment events are required and, consequently, different RNAs support a different clustering. (ii) The origin of PZSV RNA 3 is a very old duplication event that took place in the last common ancestor of the family. (iii) CiLRV RNA 3 was recruited from the last common ancestor of SpLV and EMoV and did not co-speciate with RNAs 1 and 2 of TAMV. (iv) AMV RNA 2 was recruited from the common ancestor of PNRSV and ApMV and, as such, reflects a different evolutionary history from their corresponding RNAs 1 and 3, which had indeed co-speciated. (v) The origin of APLPV RNA 1 is consistent with at least one reassortment event in which the segment was obtained from a donor related to the last common ancestor of AMV and PDV.

Recombination events during evolution of the Bromoviridae
In addition to the above-discussed independent segregation events during the diversification of the family, we looked for fingerprints of recombination events within segments as an additional source of phylogenetic noise. Table 2 shows the results of the analyses performed with the RDP program. Only recombination events significantly supported by at least three different methods have been included in the table.

Six recombination events were detected in RNA 3 (Table 2). The first three were involved in the origin of CiLRV RNA 3. The 5' and 3' ends of CiLRV were donated by TAMV (from nt 1 to 501, including the 5' UTR and the 5'-proximal part of MP, and from nt 3040 to the end of the segment, including the 3' end of CP and the 3' UTR). The region encompassing nt 1763–2026 (CP gene) of CiLRV RNA 3 was the result of a potential recombination event in which EMoV acted as the minor parental donor. In both cases, SpLV acted as the major parental donor. EMoV and SpLV share susceptible hosts in seven plant families (including Cucurbitaceae, Leguminosae and Solanaceae), whereas TAMV and SpLV also share susceptible hosts in the Leguminosae and Solanaceae. Therefore, mixed infections between these three viruses may have taken place. The fourth recombination event also involved CiLRV, SpLV and TAMV, although in this case CiLRV acted as the minor parental donor for the fragment nt 1982–2889 in the TAMV CP gene. SpLV and CiLRV only share susceptible hosts in the family Leguminosae. The fifth potential recombination event involved the 3' part of MP and the 5' part of the CP genes (nt 1198–2050) of the three bromoviruses. In this case, BMV is the recombinant virus, CCMV the major parental donor and BBMV the minor one. The opportunity for mixed infection between both parental viruses exists, as both share susceptible hosts in four plant families (Amaranthaceae, Chenopodiaceae, Leguminosae and Solanaceae). The remaining putative event involved members of different genera: the region of nt 239–505 (part of the 5' UTR and the 5' most proximal part of MP) of PSV was predicted to be donated by PNRSV, whereas the rest of its RNA 3 resembled that of TAV. Plant species from four families (Compositae, Cucurbitaceae, Leguminosae and Solanaceae) are susceptible to TAV and PNRSV.

A phylogenetic network to describe the evolution of the Bromoviridae
Fig. 3 shows the unrooted phylogenetic network obtained for the concatenated alignment of the three RNA genomic segments. This network depicts a complex evolutionary scenario in which recombination and reassortment events play a fundamental role during the diversification of the family. Remarkably, the ilarviruses constitute a homogeneous group that share only two of such exchange events with the other genera, although many potential instances of genetic exchange among species have taken place within the genus, as indicated by a highly connected network among the different members of the genus. The genera Bromovirus and Cucumovirus are clearly separated, although the amount of gene flow among their members is also high, as illustrated by the dense connectivity of the network in their neighbourhood.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Unrooted phylogenetic network describing the evolutionary history of the family Bromoviridae. The observation that the different viral species in the family are linked to each other by multiple pathways, thereby forming an interconnected network rather than a single bifurcating tree, is a consequence of recombination and reassortment events.

In this study, we were not concerned primarily with the role of selection during the genetic diversification of the different members of the family Bromoviridae. It is likely that selection has played an important role in shaping the genomes of the different viruses collectively known today as members of the family Bromoviridae. As is the case for point mutations, segment reassortment and homologous recombination create new genetic variability on which selection may operate. For example, positive selection may have been driving adaptation to new hosts, whereas negative selection may have been acting on removing genetic variability that produces deleterious effects in the host where it arose. In this study, we have shown that both segment reassortment and recombination have been frequent phenomena in the genesis of new viral species. Obviously, only those new combinations able to replicate in a given host would survive purifying selection and contribute to the next generation. In this respect, the reassortment and recombination events described here were viable ones as they all passed the filter imposed by purifying selection. Many other combinations did not succeed and hence are not detectable today. Immediately after this first selective filter, directional selection starts playing a role in which the interaction between the different parts of the newly created mosaic genomes is finely tuned. In this sense, co-evolution at the molecular level has been detected not only between amino acids belonging to the same protein but also among domains of the MP and CP proteins of PNRSV (Codoñer et al., 2006), proving that selection does not always act on individual amino acids but usually does so on the interactions between multiple amino acids or even entire domains.

	ACKNOWLEDGEMENTS

 
This study was supported by grants BFU2006-14819-C02-01/BMC (Spanish MEC-FEDER), ACOMP07/263 (Generalitat Valenciana) and the EMBO Young Investigator Program to S. F. E. F. M. C. received a fellowship from the CSIC I3P Bioinformatics programme. We thank Jennifer Commins for reading the manuscript and improving the English.

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Received 7 January 2008; accepted 13 March 2008.

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