Medical Research Council Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, UK
Correspondence
Duncan J. McGeoch
d.mcgeoch{at}vir.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The subfamily Gammaherpesvirinae is presently divided into two genera, namely Lymphocryptovirus (or 1 group) and Rhadinovirus (or
2 group). The
1 group contains EpsteinBarr virus (EBV) (see Table 1
for abbreviations of virus names) and its primate relatives, while the
2 group contains herpesviruses with hosts of many mammalian taxa; those treated in this paper have primate, rodent, and artiodactyl and perissodactyl ungulate hosts; human herpesvirus 8 (HHV8) is the single known human virus in the
2 group. Recent years have seen much activity in discovery and characterization of gammaherpesviruses. Until recently only Old World primate (OWP) viruses were known in the
1 group, but a
1 virus of a New World primate (NWP) has now been described (Callitrichine herpesvirus 3, CHV-3) (Rivailler et al., 2002a
). In the
2 group, many new viruses have been detected recently, with hosts that include primate, ungulate and carnivore species (for instance: Rovnak et al., 1998
; Greensill et al., 2000
; Lacoste et al., 2000
; Schultz et al., 2000
; Banks et al., 2002
; Kleiboeker et al., 2002
; Ehlers & Lowden, 2004
).
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METHODS |
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Computational analyses of phylogenetic trees.
The PHYLIP package (version 3.6; Felsenstein, 1989) was used to construct trees by the neighbour-joining method and to display and manipulate trees. PROTML in the MOLPHY package (Adachi & Hasegawa, 1994
) was used in preliminary maximum-likelihood (ML) evaluations of alignments of amino acid sequences and to generate lists of tree topologies. TREEADDER (McGeoch & Gatherer, 2005
) was used to comprehensively interpolate additional species into tree topologies.
For inference of phylogenetic trees, measures of divergence between amino acid sequences were in all cases made using the matrix of Jones et al. (1992). In-depth phylogenetic analyses were undertaken by two approaches. In the first, CODEML in the PAML package (version 3.14; Yang, 1997
) was used to make ML evaluations of aligned sets of amino acid sequences for sets of candidate trees, mostly with a discrete gamma distribution of eight classes of substitution rate across sites. Files output by CODEML containing log likelihoods for alignment sites were then processed by programs in the CONSEL package (version 0.1f; Shimodaira & Hasegawa, 2001
) to score trees by the approximately unbiased (AU) test and by multi-scaled bootstrap proportions (BP) (Shimodaira, 2002
).
In the second approach, Bayesian analysis using Monte Carlo Markov chains (BMCMC) was carried out on amino acid sequence alignments with MrBayes (version 3; Ronquist & Huelsenbeck, 2003), to generate posterior probability (PP) distributions of trees. Starting trees were randomly chosen and multiple runs of the program were generally made with different starting trees, to check convergence of the process. The program's defaults for prior probability settings were used. BMCMC processes incorporated a discrete gamma distribution of four classes of substitution rate across sites, included one cold and three heated chains, and were run for 250 000 or 500 000 generations. Output trees were sampled every 50 or 100 generations and typically the first 1000 trees collected were discarded to allow the process to reach stationarity.
Estimations of dates for phylogenetic events.
Dates for nodes in herpesvirus trees were derived on the basis of equating particular nodes to palaeontological dates in host lineages. Two methods were then employed. The first was to calculate by CODEML a molecular clock version of the rooted ML tree. This enforces a single rate of change across all branches, and the rate was then converted to calendar time by regression of the divergences for calibrating nodes against the calibration dates (using MINITAB). The second method used the program r8s (version 1.5; Sanderson, 2003), which aims to smooth differences in rates for branches of previously computed trees, without imposing the globally uniform rate of a molecular clock. The penalized likelihood and quasi-newtonian optimization options of the program were used.
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RESULTS |
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From considerations of tree scoring and analytical thoroughness we wished to examine sets of possible trees that were as complete as practicable. However, for feasibility in ML computations we could process exhaustively tree sets based on at most seven distinct OTUs. Virus species were therefore lumped into OTUs in cases that we regarded as uncontentious, based on previous analyses. The OTUs defined are illustrated by Fig. 1(a), which shows a tree for the 8x12 set derived as a starting point by the simple clustering method of neighbour-joining with bootstrap analysis. For ML analyses, the three
1 viruses (CHV3, EBV and RLV) were assigned to a single OTU with the NWP virus CHV3 as sister group to the two OWP viruses, the
2 OWP HHV8 and Rhesus rhadinovirus (RRV) were assigned to one OTU, as were the NWP herpesvirus ateles (HVA) and herpesvirus saimiri (HVS), and the artiodactyl Alcelaphine herpesvirus 1 (AHV1) and PLHV1. This gave a total of seven OTUs, for which 945 bifurcating unrooted trees are possible. We carried out a retrospective check on the validity of using these OTUs, to be described.
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Inspection of the trees in Fig. 1 (bg) showed that they can all be regarded as based on a constant 10-species tree (with the topology of that shown in Fig. 2
a) but with Bovine herpesvirus 4 (BHV4) and MHV4 each appearing at a variety of loci on this tree. The 10 trees in the AU set that are not included in Fig. 1
also conformed to this description. We therefore adopted, as a working hypothesis to facilitate further analysis, the view that inability to identify a single best tree reflected some property of the BHV4 and MHV4 sequences. BHV4 and MHV4 entries were then removed from the 8x12 alignment and the resulting 8x10 dataset evaluated with CODEML/CONSEL and MrBayes. This analysis gave confidence sets containing only the single tree shown in Fig. 2(a)
, and we now refer to this as the standard 10-species tree. At this point we carried out a check on the validity of our use of OTUs containing more than one species in the analysis described above: BHV4 and MHV4 were added back to the standard tree topology independently at every possible locus, to give a set of 323 trees for 12 species (so that trees with BHV4 and MHV4 within loci corresponding to the previous multiple-species OTUs were now included). This set of trees was then evaluated with CODEML/CONSEL, but no novel trees emerged in the resulting confidence sets, indicating that the use of multiple-species OTUs had been in order.
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We interpret these findings as follows. First, variability in the BHV4 locus is now seen to represent a straightforward case of very closely spaced nodes for BHV4, HHV8/RRV and HVA/HVS rather than indicating some property truly specific to BHV4. Second, we note that the results for MHV4 locus vary markedly with the analytical method. Harking back to Fig. 1(a) it can be seen that the simplest method employed, that of the neighbour-joining algorithm, gives the deepest node for MHV4, while the two CODEML analyses (Fig. 2c
) push the MHV4 node toward the HHV8/RRV clade, the more elaborate version more so. We take this correlation with analytical refinement to indicate that the MHV4 sequences possess some characteristic relative to those of the other species that requires a superior modelling process for optimal outcome, and we presume that the problem lies in an atypically high rate of substitution in MHV4 sequences as evidenced by the long terminal MHV4 branch seen in all trees. Finally, these two factors (of closely spaced nodes and idiosyncratic data) have overlapping ranges of action, so that together they acted in the analyses of Fig. 1
to obscure phylogenetic inference effectively.
The ML trees shown for the 8x12 set in Fig. 1 are, except for the low-scoring tree in part (g), compatible with treating the HHV8/RRV, HVA/HVS, BHV4 and MHV4 lineages as branching in indistinguishable order from a single multifurcation. The ML tree with this multifurcation is shown in Fig. 4
(a) (in rooted form, to be described below). We regard this as a conservative, well justified representation of the relationships among
2 lineages. For further discussion we refer to the clade containing HHV8/RRV, HVA/HVS, BHV4 and MHV4 as the MF (for multifurcated) clade. We postpone to after analysis of other alignment sets the question of whether the BMCMC analysis, with its apparently more focused confidence sets, might yield a better resolved tree.
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Each alignment was analysed by CODEML/CONSEL (with test sets of 945 trees as above) and by MrBayes. These exercises are summarized in Fig. 3 in the condensed form of majority-rule consensus trees from the BMCMC analyses. With one exception, the trees in Fig. 3
show a similar condition to those in Fig. 1
in that they have the topology of the standard 10-species tree (as defined above) plus a variety of loci for BHV4 and MHV4. The exception, Fig. 3(f)
, is for the 60K group, and here the locations of nodes for Equid herpesvirus 2 (EHV2) and HVA/HVS/BHV4 are reversed relative to the standard tree; we note, however, that this atypical configuration is associated with relatively low support in the PP distribution, and accordingly consider that the result for the 60K group can be discounted. In addition, all the trees in Fig. 3
show a long terminal branch for MHV4. We interpret these results as indicating that, despite considerable noise, the smaller datasets show general consistency with the 8x12 set both in tree topology and in uniquely high level of substitution for the MHV4 terminal branch, so that these features should be taken as conserved across the genomes.
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CODEML was used to compute ML branch lengths for the quadrifurcated topology, for all three datasets. The lengths of corresponding branches in these 21x12 and 28x11 trees were compared to those of the 8x12 tree. In both cases proportions were found to be closely maintained across the trees; relative to the 8x12 tree branch lengths in the 21x12 and 28x11 trees are expanded by overall factors of 1·21 and 1·25, respectively (estimated by regression). Thus the 8x12 set, versions of which have previously been used to examine phylogeny across the Herpesviridae, gives a representation of relationships that is consistent within the Gammaherpesvirinae with that from the largest accessible gene sets.
The trees discussed so far are unrooted. From previous phylogenetic analyses that included data from the Alpha-, Beta- and Gammaherpesvirinae we know that the root for the gammaherpesvirus tree lies between the 1 and
2 groups (McGeoch et al., 1995
, 2000
). When we compared the 8x12 ML quadrifurcated tree (Fig. 4a
) with the rooted gammaherpesvirus portion of a ML tree for all three subfamilies based on sequences from seven genes and one partial gene of 20 species (data from McGeoch et al., 2000
), we observed a close proportionality between pairs of corresponding branch lengths, as illustrated in Fig. 4(b)
. This overall conservation in proportions then allowed transfer of the root position to the 8x12 tree with good precision, as indicated in Fig. 4(a)
. With the addition of the root locus, Fig. 4(a)
gives our current optimal representation of gammaherpesvirus phylogeny.
Coevolution of host and gammaherpesvirus lineages
As mentioned, mammalian herpesvirus lineages show extensive signs of apparent coevolution with host lineages. Interpretation on this basis has been least satisfactory for the Gammaherpesvirinae, in part because of uncertainties in the gammaherpesvirus tree (McGeoch et al., 2000; McGeoch, 2001
). We revisited this topic in light of our now robust assignment of major lineages in the tree, to examine what correlations of branching pattern and branch proportions could be made between the host and virus trees, guided by criteria of parsimony and generality. We have identified a scheme that provides an economical account of major features of the gammaherpesvirus tree in terms of hostvirus coevolution, and is also consistent with features in the trees for alpha- and betaherpesviruses.
Fig. 5(a) depicts a tree for the higher taxa of relevant host groups, with timescale [as millions of years before the present (Ma)] based primarily on Springer et al. (2003)
, and Fig. 5(b)
presents the gammaherpesvirus tree in a format intended to emphasize proposed correspondences with the host tree. We interpret the following as being of co-evolutionary origin: the OWP and NWP
1 lineage, the ruminant AHV1 and suid PLHV1 lineages, and the perissodactyl EHV2 lineage. Each of these are known to be populated, beyond the few species represented in our analyses, with other viruses that have hosts in the same group (Rovnak et al., 1998
; Ehlers et al., 1999
, 2003
; Kleiboeker et al., 2002
). The only portion of the tree in Fig. 4(a)
not thus accounted for is the MF clade, and we interpret this as having a non-coevolutionary origin, involving transfer between host species. The OWP and NWP
2 virus lineages could, however, have arisen in a co-evolutionary mode during subsequent development of the MF clade. Treating the MF clade as non-coevolutionary in origin efficiently rationalises a number of features, namely: the position of its origin in the tree; the existence of two groupings of primate gammaherpesviruses; the occurrence of BHV4 in a separate location from other ruminant viruses; and the non-correspondence between the location of MHV4 and the location in the host tree of the rodent lineage (shown in Fig. 5a
as a grey line).
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DISCUSSION |
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At the end of these exhaustive analyses, the gammaherpesvirus tree obtained is completely compatible with that from our previous work (McGeoch et al., 2000; McGeoch, 2001
), with the addition of four virus species, with assurance of robustness and generality, and with improved insight into underlying complications. Disentangling the phylogeny of the
2 group presented difficulties, and recognizing that the nodes for HHV8/RRV, HVA/HVS and BHV4 are effectively coincident and that there is a special problem with the locus of MHV4 were significant steps toward understanding relationships. The overall phylogeny appears to be constant across the genomes, but trees based on small sets of genes were generally noisy and the larger datasets were required for optimal resolution. With the overall constancy of tree topology and proportions seen with the large datasets, we are confident that the optimal tree as presented in Fig. 4(a)
is well founded. We regard the tree as comprising four major clades: the
1 group, the AHV1/PLHV1 lineage, the EHV2 lineage and the MF clade. There are limited sequence data available for many other gammaherpesviruses, but we know of none that demonstrably falls outside these clades.
It is clear that MHV4 sequences have been accumulating changes atypically fast. This effect is not seen with another rodent herpesvirus, the betaherpesvirus murine cytomegalovirus (McGeoch et al., 2000). The visible relative rate of change for MHV4 is, of course, an average over the time since the MHV4 lineage diverged from its sister groups. We cannot, with available data, study substitution characteristics of MHV4 DNA directly; two genome sequences for MHV4 are available (Table 1
), but they are of the same strain and very close to identical (Nash et al., 2001
) so their comparison is not useful. It is likely that the effect has applied across the genome: it is not a matter of selected mutation in one or a few loci, such as seen with the K1 gene of HHV8 (McGeoch, 2001
). The effect could result from relaxed stringency in genomic replication, from more frequent cycles of replication than with other viruses of the group, or from larger virus populations (with ongoing recombination). Whatever its mechanism, this phenomenon may reflect some significant underlying difference between MHV4's biology and those of other
2 herpesviruses.
Our date estimates for the gammaherpesvirus tree based on hostvirus coevolution (Table 3) are likely to be more precise for
2 than for
1 nodes with the calibration system applied. In the
1 clade, it has previously been apparent that EBV and RLV may be more closely related than the 23 Ma date expected for cospeciation (Ehlers et al., 2003
; Gerner et al., 2004
). Conversely, in the
2 group, HHV8 and RRV are more distant than cospeciational counterparts (Greensill et al., 2000
; Schultz et al., 2000
). These examples emphasize that the concept of hostherpesvirus coevolution should not be pursued down to the species level. If the coevolution hypothesis is valid for higher taxa and longer timescales, then the estimates indicate a considerable antiquity for development of the MF clade, close to the end of the Cretaceous (65 Ma). We presume that the MF clade originated by transfer of a virus between host species. The clade's immediate ancestor could have been a virus with a perissodactyl or carnivore host (see location of the carnivore lineage in the host tree, shown in Fig. 5a
as a grey line). Involvement of a carnivore gammaherpesvirus ancestor would be consistent with our estimates for the development of the MF clade (Table 3
) in relation to that for divergence of perissodactyl and carnivore lines (80·4 Ma; Springer et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 6 September 2004;
accepted 1 November 2004.