A performance study of the impact of recombination on species tree analysis

Background

The most widely used state-of-the-art methods for reconstructing species phylogenies from genomic sequence data assume that sampled loci are identically and independently distributed. In principle, free recombination between loci and a lack of intra-locus recombination are necessary to satisfy this assumption. Few studies have quantified the practical impact of recombination on species tree inference methods, and even fewer have used genomic sequence data for this purpose. One prominent exception is the 2012 study of Lanier and Knowles. A main finding from the study was that species tree inference methods are relatively robust to intra-locus recombination, assuming free recombination between loci. The latter assumption means that the open question regarding the impact of recombination on species tree analysis is not fully resolved.

Results

The goal of this study is to further investigate this open question. Using simulations based upon the multi-species coalescent-with-recombination model as well as empirical datasets, we compared common pipeline-based techniques for inferring species phylogenies. The simulation conditions included a range of dataset sizes and several choices for recombination rate which was either uniform across loci or incorporated recombination hotspots. We found that pipelines which explicitly utilize inferred recombination breakpoints to delineate recombination-free intervals result in greater accuracy compared to widely used alternatives that preprocess sequences based upon linkage disequilibrium decay. Furthermore, the use of a relatively simple approach for recombination breakpoint inference does not degrade the accuracy of downstream species tree inference compared to more accurate alternatives.

Conclusions

Our findings clarify the impact of recombination upon current phylogenomic pipelines for species tree inference. Pipeline-based approaches which utilize inferred recombination breakpoints to densely sample loci across genomic sequences can tolerate intra-locus recombination and violations of the assumption of free recombination between loci.

     

Molecular analysis of the recombination junctions of lambda bio transducing phages.

Summary To examine the mechanism of recombination involved in the formation of specialized transducing phage during the induction of bacteriophage we have determined the nucleotide sequences of the recombination junctions of bio phages. The results indicate that abnormal excision takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology (5–14 bp). Some of the sequences of the recombination sites were similar to the consensus sequences of DNA gyrase-cleavage sites and repetitive extragenic palindromic (REP) sequences. These results showed that abnormal excision is a type of illegitimate recombination. The possible involvement of DNA gyrase in this recombination is discussed.     

Deletion breakpoints associated with the Prader-Willi and Angelman syndromes (15q11-q13) are not sites of high homologous recombination

Deletions of 15q11.2-q12 are associated with either the Prader-Willi (PWS) or Angelman (AS) syndromes. It has been suggested that excessive recombination in this region might explain the high frequency of such deletions, and the frequent involvement of chromosome 15 in translocations and nondisjunction. We have studied recombination in the PWS region by linkage analysis of non-PWS families. No recombination was found (with maximum lod scores greater than 3.0) for most pairwise combinations of probes: 39, IR4-3R, ML34, 189-1, 3-21. A hotspot of recombination is observed between loci detected by p3-21 and pIR10-1. The female recombination fraction in this region was significantly higher than that for males. Close linkage with 0.06 recombination was found for the IR10-1 and CMW-1 pair. No excess recombination was found between sites bounding common breakpoints observed in deletions associated with PWS and AS. It is suggested that these deletions form frequently because of the presence of duplicated DNA sequences and/or inversions in this region, and not because of a high rate of homologous recombination.     

Recombination and Replication

The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.The interplay between replication and recombination is complex in terms of both mechanism and integration within DNA metabolism. At the heart of this interplay is the requirement for single-stranded DNA (ssDNA), the substrate for DNA-strand-exchange proteins, to initiate recombination (Cox 2007b; San Filippo et al. 2008). Whether, when, and where this ssDNA is generated determines the functional relationship between replication and recombination, a relationship that can operate in both directions. Homologous recombination enzymes are critical for successful completion of genome duplication (Kogoma 1997; Cox et al. 2000) but DNA replication also underpins homologous recombination, as discussed elsewhere in this collection. The links between recombination and replication are therefore intimate and one cannot be considered in isolation from the other. However, involvement of DNA-strand-exchange proteins, regardless of the metabolic context, comes with the unavoidable risk of genome rearrangements. This genome instability can occasionally increase evolutionary fitness but more frequently is deleterious to the viability of the individual.This review will focus on fundamental aspects of the links between replication and recombination enzymes rather than simply providing a list of known enzymes and reactions. The substrate, DNA, is identical in all of these reactions and this is reflected in the high mechanistic conservation of replication and recombination.     

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda

Summary To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage and pBR322 derivatives containing DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA ⁺ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of -plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.     

Pairing for Recombination in Lgv of Caenorhabditis Elegans: A Model Based on Recombination in Deficiency Heterozygotes

The effect of deficiencies on recombination was studied in Caenorhabditis elegans. Heterozygous deficiencies in the left half of linkage group V [LGV(left)] were shown to inhibit recombination to their right. Fourteen deficiencies, all to the left of unc-46, were analyzed for their effect on recombination along LGV. The deficiencies fell into two groups: 10 "major inhibitors" which reduce recombination to less than 11% of the expected rate between themselves and unc-46; and four "minor inhibitors" which reduce recombination, but to a much lesser extent. All four minor inhibitors delete the left-most known gene on the chromosome, while six of the ten major inhibitors do not (i.e., these are "internal" deficiencies). Where recombination could be measured on both sides of a deficiency, recombination was inhibited to the right but not to the left. In order to explain these results we have erected a model for the manner in which pairing for recombination takes place. In doing so, we identify a new region of LGV, near the left terminus, that is important for the pairing process.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Identification of Recombinant Human Rhinovirus A and C in Circulating Strains from Upper and Lower Respiratory Infections

Human rhinoviruses (HRVs), in the Enterovirus genus within the family Picornaviridae, are a highly prevalent cause of acute respiratory infection (ARI). Enteroviruses are genetically highly variable, and recombination between serotypes is known to be a major contribution to their diversity. Recently it was reported that recombination events in HRVs cause the diversity of HRV-C. This study analyzed parts of the viral genes spanning the 5′ non- coding region (NCR) through to the viral protein (VP) encoding sequences of 105 HRV field isolates from 51 outpatient cases of Acute Respiratory Infectious Network (ARINET) and 54 inpatient cases of severe lower respiratory infection (SLRI) surveillance, in order to identify recombination in field samples. When analyzing parts of the 5′NCR and VP4/VP2 encoding sequences, we found intra- and interspecies recombinants in field strains of HRV-A and -C. Nineteen cases of recombination events (18.1%) were found among 105 field strains. For HRV-A, there were five cases (4.8%) of intraspecies recombination events and three cases (2.8%) of interspecies recombination events. For HRV-C, there were four cases (3.8%) of intraspecies recombination events and seven cases (6.7%) of interspecies recombination events. Recombination events were significantly more frequently observed in the ARINET samples (18 cases) than in the SLRI samples (1 case; P< 0.0001). The recombination breakpoints were located in nucleotides (nt) 472–554, which comprise stem-loop 5 in the internal ribosomal entry site (IRES), based on the HRV-B 35 sequence (accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ445187","term_id":"217316504","term_text":"FJ445187"}}FJ445187). Our findings regarding genomic recombination in circulating HRV-A and -C strains suggest that recombination might play a role in HRV fitness and could be a possible determinant of disease severity caused by various HRV infections in patients with ARI.     

The Evolutionary Advantage of Recombination. II. Individual Selection for Recombination

Based on the Fisher-Muller theory of the evolution of recombination, an argument can be constructed predicting that a recessive allele favoring recombination will be favored, if there are either favorable or deleterious mutants occurring at other loci. In this case there is no clear distinction between individual and group selection. Computer simulation of populations segregating for recessive or dominant recombination alleles showed selection favoring recombination, except in the case of a dominant recombination allele with deleterious background mutants. The relationship of this work to parallel investigations by Williams and by Strobeck, Maynard Smith, and Charlesworth is explored. All seem to rely on the same phenomenon. There seems no reason to assume that the evolution of recombination must have occurred by group selection.     

Biochemical control of recombination in Saccharomyces cerevisiae. I. L-histidine inhibition of intragenic mitotic recombination at the ad 3 locus

Summary A yeast strain heteroallelic at the ad ₃ locus is used to study mitotic intragenic recombination. L-histidine inhibits the recombination at this locus in strains heteroallelic for all possible combinations between the four alleles studied. No other amino acid has this effect. The kinetic of recombination was studied by addition of L-histidine at different times or by compeating the L-histidine present with D-histidine added at different times. The two techniques gave similar results showing that the recombination takes place between the 8th and 24th hour after plating although it is expressed a few days later.Taking advantage of the early interval in which the recombination takes place and of the fact than the petite mutation is induced by acriflavine only in new formed buds, we developed a technique to study the recombination in liquid medium, thanks to which, we were able to show that L-histidine inhibits the genetic event itself.     

Mapping dystrophin gene recombinants in Greek DMD/BMD families: low recombination frequencies in the STR region

A systematic study of 42 Greek DMD/BMD families using 14 polymorphic markers that span the dystrophin gene was performed in order to assess the position and frequency of recombinants in the Greek population and to test whether hot spots of recombination and deletions coincide when exclusively studying DMD/BMD families. We report a low percentage of recombination between markers STR44 and STR50; otherwise, the distribution of recombination events in other parts of the gene is largely in agreement with previously published data on Centre d'Etude du Polymorphisme Humaine families. We therefore propose that recombination frequencies and the correlation between recombination and deletion hot spots should be evaluated on DMD/BMD families exclusively.     

Homology is not required for recombination mediated by DNA gyrase of Escherichia coli

Summary We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al. 1982). In the present paper, we have studied the nucleotide sequences of several recombination junctions of -pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the and pBR322 recombination sites. Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination. Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple crossovers in our in vitro system.     

Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'''' of Fission Yeast

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.     

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.RNA viruses are able to undergo rapid genetic changes in order to adapt to new hosts or environments. Although much of this flexibility is due to the error-prone nature of the RNA-dependent RNA polymerase, which generates an array of different point mutations within the viral population (23), recombination is also a common and important mechanism for generating viral diversity (18, 31, 42, 58). Recombination occurs when the RNA-dependent RNA polymerase switches templates during replication, an event that is favored when both templates share identical or very similar sequences. Three types of RNA recombination have been identified: homologous recombination occurs at sites with exact sequence matches; aberrant homologous recombination requires sequence homology, but crossover occurs either upstream or downstream of the site of homology, resulting in a duplication or deletion; and nonhomologous (or illegitimate) recombination is independent of sequence homology (31, 42).When the same cell is infected by viruses of two different strains, or even different species, recombination between their genomic RNAs can potentially lead to the emergence of new pathogens. A case in point is the emergence of Western equine encephalitis virus, a member of the genus Alphavirus, family Togaviridae, which arose by homologous recombination between Eastern equine encephalitis virus and Sindbis virus (14).Some mammalian RNA viruses can recombine at a frequency that is detectable in experimental settings (1, 2, 55), and phylogenetic analysis of partial or complete genome sequences suggests that RNA recombination is a widespread phenomenon. Naturally occurring recombinant viruses have been identified in almost every family of positive-stranded RNA viruses (31, 58).Flaviviruses are members of the genus Flavivirus, family Flaviviridae, a family that also includes the genera Pestivirus and Hepacivirus. Several of the flaviviruses are important human pathogens, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), the dengue viruses, yellow fever virus, and tick-borne encephalitis virus (TBEV).Although there has never been a report of a pathogenic flavivirus strain arising due to recombination involving attenuated vaccine strains (39), the urgent necessity to develop tetravalent vaccines containing all four serotypes of dengue virus—two such vaccines are currently undergoing clinical testing (45)—has recently brought the recombination issue to the forefront of discussion among researchers, regulators, and vaccine producers (39). It has been suggested that recombination, either between the strains present in a multivalent vaccine or between an attenuated vaccine strain and a wild-type strain, could lead to the emergence of new viruses with unpredictable properties (49).So far, recombination between flavivirus genomes has not been demonstrated directly in the laboratory. However, phylogenetic analysis of partial genome sequences available in the GenBank database has suggested that homologous recombination may have occurred between closely related strains of dengue virus (20, 52, 54, 59). An experimental approach for assessing the ability of flavivirus genomes to recombine is therefore urgently needed.Flavivirus virions are composed of a single-stranded, positive-sense RNA genome that, together with the capsid protein C, forms the viral nucleocapsid. The nucleocapsid is covered by a lipid envelope containing the surface glycoproteins prM and E. These glycoproteins drive budding at the membrane of the endoplasmic reticulum during the assembly stage and mediate entry of the virus into host cells (41). Replicons, defined as self-replicating, noninfectious RNA molecules, can be generated by deleting parts or all of the region coding for the structural proteins C, prM, and E from the viral genome but maintaining all seven of the nonstructural proteins and the flanking noncoding sequences, which are required in cis for RNA replication (25). By providing the missing structural protein components in trans, replicons can be packaged into virus-like particles that are capable of a single round of infection (10, 15, 24, 47).Typically, researchers developing novel replicating vaccines, especially ones that involve multiple components, make an effort to come up with strategies to prevent recombination, for example by “wobbling” codons, i.e., replacing codons in homologous regions with synonymous ones encoding the same amino acid but consisting of a different nucleotide triplet (50, 57). In this study, in order to assess the propensity of flavivirus genomes to recombine, we took an opposite approach, establishing a “recombination trap” that favors the selection and sensitive detection of recombination products. This system takes advantage of the ability of replicon pairs containing deletions in their structural protein genes to complement each other in trans and thus be propagated together in cell culture, and by passage at limiting dilutions, it allows infectious RNA genomes arising by recombination between the two replicons to be preferentially selected.Using the recombination trap, we have now obtained the first direct evidence of recombination between flavivirus genomes in the laboratory. Aberrant homologous recombination was observed twice with JEV replicons, resulting in viruses with unnatural gene arrangements and reduced growth properties compared to those of wild-type JEV. No infectious recombinants of any kind were obtained when TBEV or WNV replicons were used. Interestingly, we never detected a fully infectious wild-type genome arising by homologous recombination in any of these systems. The results of this study show that the propensity of flavivirus genomes to recombine in the region coding for the structural proteins appears to be quite low, suggesting that recombination does not represent a major risk in the use of live, attenuated flavivirus vaccines.     

Multiple Interactions of a DNA-Binding Protein IN VIVO. III. Phage T4 Gene-32 Mutations Differentially Affect Insertion-Type Recombination and Membrane Properties

We have investigated the in vivo roles of T4 gene- 32 protein in recombination. We have studied the effects of gene- 32 mutations under conditions that allow normal DNA replication and are permissive for progeny production. Under these conditions, certain gene- 32 mutations specifically reduce insertion-type (short-interval) recombination but none affect crossover-type (long-interval) recombination (see Figure 5). Heterozygote frequencies in all gene-32 mutants are similar to or higher than in a gene-32⁺ background and are not correlated with recombination deficiencies. "Recombination-deficient" alleles are dominant or codominant over the "recombination-proficient" gene-32 mutation tsL171. This explains apparent discrepancies between a gene-32 map deduced from two-factor crosses and the map derived from three-factor crosses.

We have also found that the "recombination proficient" mutation tsL171 and its homoalleles suppress the characteristic plaque morphology of rII mutants. Under restrictive conditions, tsL171 is partially suppressed by rII mutations, which allow the use of host ligase in recombination.

Our present and previous results are discussed in terms of current recombination models. We conclude that gene-32 protein functions in recombination by forming a complex with DNA, with recombination enzymes and with membrane components. Since gene-32 protein interacts with many components of this recombination complex, gene-32 mutations may differentially affect various recombination steps.

     

Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.     

Experimental evolution of recombination and crossover interference in <Emphasis Type="Italic">Drosophila</Emphasis> caused by directional selection for stress-related traits

Background

Population genetics predicts that tight linkage between new and/or pre-existing beneficial and deleterious alleles should decrease the efficiency of natural selection in finite populations. By decoupling beneficial and deleterious alleles and facilitating the combination of beneficial alleles, recombination accelerates the formation of high-fitness genotypes. This may impose indirect selection for increased recombination. Despite the progress in theoretical understanding, interplay between recombination and selection remains a controversial issue in evolutionary biology. Even less satisfactory is the situation with crossover interference, which is a deviation of double-crossover frequency in a pair of adjacent intervals from the product of recombination rates in the two intervals expected on the assumption of crossover independence. Here, we report substantial changes in recombination and interference in three long-term directional selection experiments with Drosophila melanogaster: for desiccation (~50 generations), hypoxia, and hyperoxia tolerance (>200 generations each).

Results

For all three experiments, we found a high interval-specific increase of recombination frequencies in selection lines (up to 40–50 % per interval) compared to the control lines. We also discovered a profound effect of selection on interference as expressed by an increased frequency of double crossovers in selection lines. Our results show that changes in interference are not necessarily coupled with increased recombination.

Conclusions

Our results support the theoretical predictions that adaptation to a new environment can promote evolution toward higher recombination. Moreover, this is the first evidence of selection for different recombination-unrelated traits potentially leading, not only to evolution toward increased crossover rates, but also to changes in crossover interference, one of the fundamental features of recombination.