Genetic exchange between organisms, called recombination, occurs in all biological kingdoms and is also common in viruses in which it may threaten the long-term control of important human pathogens such as HIV and influenza. Although recombination can produce advantageous gene combinations, bioinformatic analyses of bacterial genomes have suggested that recombination is not well tolerated when it involves exchanges of genes that interact with a lot of other genes. Using laboratory-constructed recombinants of a small plant virus called MSV, Martin and co-workers provide the first direct experimental evidence that the evolutionary value of exchanging a genome fragment is constrained by the number of ways in which the fragment interacts with the rest of the genome. They note that fitness losses suffered by artificial MSV recombinants increase with decreasing parental relatedness. Furthermore, these losses accurately anticipate the patterns of genetic exchange detectable in natural MSV recombinants, suggesting that they accurately reflect the impact of deleterious selection on natural isolates of the virus.

Genetic recombination may predate the evolution of cellular life [1] and is the basis of ubiquitous biological processes such as DNA repair and sexual reproduction. The combinatorial nature of recombination can provide organisms with vastly more evolutionary options than are available through mutation alone [2–4]. However, kingdom-wide analyses of bacterial recombination [5] and DNA-shuffling studies [6,7] have indicated that the evolutionary value of recombination can vary depending on both the genes and the sub-gene modules transferred. In bacteria, the complexity hypothesis has been proposed to explain an imbalance in detectable informational and operational gene transfers between species [5]. Similarly, the schema hypothesis has been proposed to explain patterns of sequence mosaics observed in DNA-shuffling experiments [7]. Although the complexity hypothesis concerns genes within the context of genomes, the schema hypothesis concerns amino acids within the context of proteins. Both hypotheses are conceptually related and propose that the functionality of sequence fragments in foreign genetic backgrounds is inversely correlated with the complexity of interaction networks within which they must function.

Here we provide experimental support for these hypotheses using the small single stranded plant DNA virus, Maize streak virus (MSV; Geminiviridae, Mastrevirus) as a model organism to investigate the effect of genomic recombination on viral fitness.

Figure 1 Laboratory Constructed MSV Recombinants and Their Relative Fitness (as Measured by ICLAs) in Maize

As a correlate of viral fitness, we determined the induced chlorotic leaf areas (ICLA) of parental and recombinant viruses in infected maize plants [8]. The relationship between symptom severity and fitness is complex for most pathogens. However, MSV populates mesophyll cells within precisely defined chlorotic lesions of infected maize leaves [9] and the chlorotic surface area of an infected leaf is positively correlated with the total amount of viral DNA within the leaf [10,11]. The correlation between MSV pathogenicity and fitness is also evident in the greater geographical distribution and incidence of more pathogenic MSV genotypes relative to less pathogenic genotypes [12].

For each pair of reciprocal recombinants, we defined the recombination tolerance index (Ti) as the average ICLA of the recombinant pair divided by the average ICLA of their parental viruses. If reciprocally exchanged sequence fragments continue to function as well within recombinant genomes as they did in their original genomic backgrounds, we expect that the average ICLA of reciprocal pairs should be identical to that of their parental pairs—i.e., we would expect a Ti = 1.0. Conversely a drop in Ti below 1.0 would indicate that reciprocally exchanged sequences might not function as well in their new genomic backgrounds as they did in their original backgrounds.

In 17 out of 18 recombination experiments, the average ICLA of reciprocal recombinant pairs was lower than that of their parental viruses (i.e., Ti < 1). Values of Ti generally decreased with increasing divergence of exchanged sequences, with a distinct rate of decrease for each genome region exchanged (Figure 2). Given equal degrees of divergence, it seems that the short intergenic region (SIR) and movement protein gene (MP) function better in foreign genetic backgrounds than do the replication-associated protein gene (Rep), the coat protein gene (CP), or the long intergenic region (LIR). In other words, the SIR and MP appear more modular than the other regions of the genome.

Figure 2 Tolerance of Recombination in MSV Differs According to the Genome Region Involved and the Degree of Divergence between Exchanged Sequences

Figure 3 The Network of Known Protein–Protein and Protein–DNA Interactions during a MSV Infection of Maize

To determine whether the known network of interactions occurring during an MSV infection is anticipated by our Ti data, it was necessary to first extract a relative modularity score for each of the MSV genome regions from the Ti plots. To do this we fitted quadratic equations to the plots (see Figure 2) to estimate similarly tolerable degrees of recombination-induced diversification (RID) in the five genomic regions. Our aim in fitting a line to the Ti plots was to objectively estimate degrees of RID tolerated in the different genome regions at particular Ti values. For example, fitting quadratics to the plots and picking a Ti value of 0.9 (reciprocal recombinants have an average ICLA 90% that of their parents) the corresponding estimates of tolerable recombination induced diversification in the SIR, MP, CP, LIR, and Rep are 15.3%, 8.3%, 2.7%, 3.3%, and 3.8% respectively. To avoid any biases due to “cherry picking” the Ti values we used to compare the different genome regions, we examined estimated recombination tolerances over the range of Ti values between 0.99 and 0.7. There is good correlation between the number of known MSV intergenome component protein–protein and protein–DNA interactions for different genome regions (SIR = 1, MP = 2, CP = 5, LIR = 4, and Rep = 4) and recombination tolerance estimated for these same regions over the entire Ti range between 0.94 and 0.7 (Pearson's R² > 0.87, p < 0.05, Spearman's rho corrected for ties = −0.975, p = 0.051).

Table 1 Evidence of Recombination within Five Defined Regions of Publicly Available African Streak Virus Genome Sequences

Figure 4 The Experimentally Determined Relative Recombination Tolerance of Five MSV Genomic Regions Is Highly Correlated with Values Inferred from Nature

By demonstrating a negative correlation between the relative modularity of defined genomic regions and the complexity of interactions in which they are involved we have provided experimental support for the complexity hypothesis [5]. This hypothesis was proposed to explain the disparity in informational (those involved in transcription, translation, and related processes) and operational (those involved in housekeeping) gene transfer rates in bacteria. It states that because informational genes are generally involved in more complex interactions than operational genes, they are less likely to continue functioning well after horizontal transfer.

The progressive decrease in tolerance of recombination with increasing divergence of exchanged sequences observed in Figure 2 has strong parallels with parental sequence imbalances observed in “family shuffling” variants of DNA-shuffling experiments [2]. The functional genes produced by shuffling three or more distinct sequences (i.e., 60%–85% identical) are usually derived predominantly from either one sequence or combinations of the most similar sequences [2,6,23,24]. The schema hypothesis proposes that these imbalances are due to the probability of recombinant protein-fold disruption increasing with increasing divergence of parental sequences [6,7]. We shuffled entire intergenic regions and genes, and therefore the effect we observed cannot be explained directly in terms of the schema hypothesis. We have, however, provided empirical evidence for a whole-genome analogue of this hypothesis: The probability of the normal network of intragenome interactions being disrupted by recombination increases with increasing divergence of the exchanged fragments.

The successful inheritance of genomic fragments through recombination is expected to depend on the maintenance of important intragenome interactions. After all, the exchange of a genome fragment could be seen as a simultaneous introduction of multiple mutations. Negative or purifying selection should remove those recombinants that break the epistatic interactions that define the architecture of a particular genome, whereas genetic drift might permit the survival and spread of “neutral” recombinants. Alternatively, positive selection should favour the spread of rare recombinants with improved genomic interactions. The genomic interactions in the (natural) parental viruses used in these experiments have most likely been optimised through selection over long evolutionary periods. None of the recombinants generated from these viruses was more fit than the fitter of its parents, which is expected if negative selection is the dominant force that now maintains the integrity of these genomes.

The relative degree of modularity that we demonstrated experimentally for each genome component appears to be reflected in the recombination events detected within the same regions in natural viruses represented in GenBank. This correlation is surprising because the natural recombinants—unlike the recombinants we constructed in the laboratory—involve exchanges of fragments of genes or intergenic regions. Such exchanges may disrupt intraprotein or intra-intergenic region interactions as well as interaction networks amongst whole genes and intergenic regions. Survival of the natural viruses with detectable recombination events in coding regions presumably depended on their inheritance of sequences that did not overly disrupt either intraprotein or intergene/intergenic region interaction networks; survival of natural intergenic region recombinants and those we generated in the laboratory would have been subject only to the latter constraint. The correlation between our experimental results and the inferred natural recombinants may indicate that maintenance of intergenome component interactions is the principal determinant of recombination tolerance (at least for MSV and closely related viruses). Alternatively, a requirement for the preservation of both intergenome component, and intragene, interaction networks has a net effect that is difficult to distinguish from either constraint operating alone.

We have provided experimental support for the complexity hypothesis by demonstrating a relationship between the relative modularity of defined genomic regions and the complexity of interactions in which they are involved. The striking correlation between our experimental results and the types of recombination observed in nature lends credence to the notion that these detectable modularity differences are evolutionarily relevant. Our results also suggest that the degree of similarity between an inherited sequence and the sequence it replaces is an important additional determinant of recombinant fitness. Whereas recombination can substantially increase the evolutionary options of an organism, the obligatory maintenance of co-evolved interaction networks may severely restrict its evolutionary value.

We would like to thank the South African National Research Foundation and National Bioinformatics Network for funding this work. DP is supported by the Ramón y Cajal Programme of the Spanish Government, and funded by grants R01-GM55276 from the United States National Institutes of Health, and BFU2004–02700 from the Spanish Education and Science Ministry (MEC).