Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on June 15, 2004

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Human Molecular Genetics, 2004, Vol. 13, No. 16 1785-1791
DOI: 10.1093/hmg/ddh183
Human Molecular Genetics, Vol. 13, No. 16 © Oxford University Press 2004; all rights reserved

Rapid evolution of primate antiviral enzyme APOBEC3G

Jianzhi Zhang^* and David M. Webb

Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA

Received April 23, 2004; Accepted June 3, 2004

DDBJ/EMBL/GenBank accession nosAY639867–AY639869

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human cytidine deaminase APOBEC3G and the virion infectivity factor (vif) of the human immunodeficiency virus (HIV) are a pair of antagonistic molecules. In the absence of vif, APOBEC3G induces a high rate of dC to dU mutations in the nascent reverse transcripts of HIV that leads to the degradation of the HIV genome. HIV vif, on the other hand, can suppress the translation and trigger the degradation of human APOBEC3G. Here, we studied the rate of APOBEC3G gene evolution from five hominoids and two Old World monkeys. Averaged across the entire coding region, the rate of non-synonymous nucleotide substitutions is 1.4 times the rate of synonymous substitutions, strongly suggesting that APOBEC3G has been under positive Darwinian selection. A comparison between the nucleotide polymorphisms within humans and the substitutions among the seven primates reveals a significant excess of non-synonymous substitutions. Furthermore, the rate of charge-altering non-synonymous substitution is 1.8 times that of charge-conserving substitution, indicating that the selection is promoting the diversity of the protein charge profile. However, no difference in selective pressure on APOBEC3G is detected between hosts and non-hosts of HIV or simian immunodeficiency virus (SIV). These results, together with recent findings that the antiviral activity of APOBEC3G is not limited to HIV/SIV, suggest that the selective pressure on APOBEC3G is not solely from HIV/SIV and that APOBEC3G is a broad antiviral enzyme. The identification of pervasive positive selection for charge-altering amino acid substitutions supports the hypothesis of electrostatic interactions between APOBEC3G and vif or its functional equivalents.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Strategies to mitigate our susceptibility to the human immunodeficiency virus (HIV) can be informed by knowledge of how genes involved in the human–HIV interaction evolved. Recent findings of antagonism between human APOBEC3G protein and the virion infectivity factor (vif) of HIV shed new light on this important topic. When the vif gene is deleted, HIV can replicate only in certain human cells termed permissive cells, but not in other cells termed non-permissive cells (1,2) due to the presence of an inhibitory factor (3,4). Using cDNA subtraction, Sheehy et al. (5) cloned the inhibitory factor gene CEM15 from the non-permissive cell line CEM. They demonstrated that transient or stable expression of CEM15 in permissive cells leads to the non-permissive phenotype, and that this phenotype is overcome by the presence of vif. CEM15 was found to be homologous to the RNA editing enzyme APOBEC1 (apoB editing catalytic subunit 1), which performs C-to-U editing in apolipoprotein B (apoB) messenger RNA (mRNA) (5). Subsequently, CEM15 was renamed APOBEC3G.

After infecting human cells, the RNA genome of HIV is reverse transcribed. In the absence of vif, APOBEC3G induces deamination of dC in the first strand of cDNA, resulting in hypermutation of dC to dU (6–9). It has been proposed that uracil N-glycosylase (UNG) then removes dU in the DNA, leading to its degradation (10,11). Alternatively, if dU escapes from UNG, the mutations are incorporated into the RNA genome of HIV and appear as high incidences of G-to-A mutations. These mutations may disrupt open reading frames and render HIV non-infectious. Thus, without vif, the infectivity of HIV is destroyed. vif, however, can overcome the action of APOBEC3G by suppressing the translation of APOBEC3G and inducing its degradation by a proteasome-dependent pathway (12–16). It has been shown that vif physically interacts with APOBEC3G and other cellular proteins to trigger the ubiquitination and degradation of APOBEC3G (12–15,17). The interaction of vif and APOBEC3G is partially species-specific. For instance, HIV vif apparently counteracts the APOBEC3G of the human and chimpanzee, but not that from the mouse or African green monkey (18). These observations suggest that the host APOBEC3G and viral vif protein may engage in antagonistic coevolution, which will result in rapid amino acid substitutions in both the proteins [i.e. the red-queen hypothesis (19)]. Many HIV genes, including vif, have been shown to evolve rapidly by diversifying positive selection (20). Here, we test whether the primate APOBEC3G gene also evolves under positive selection and examine whether the selective pressure comes from its antagonism with vif of the HIV/SIV (simian immunodeficiency virus).

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Phylogeny of the primate APOBEC3G gene sequences
We sequenced the complete coding region of the APOBEC3G gene from the pygmy chimpanzee, gorilla and orangutan, and downloaded from the GenBank the sequences for human, chimpanzee, macaque and African green monkey. Figure 1 shows the alignment of the seven primate APOBEC3G protein sequences. Because APOBEC3G belongs to an Anthropoid-specific gene family (APOBEC3) that contains multiple functional genes and pseudogenes (21), it is important to verify that the obtained sequences are orthologous. For this, we constructed a protein neighbor-joining tree of the seven primate APOBEC3G genes, together with human APOBEC3B, human APOBEC3F and mouse APOBEC3 (CEM15). We included APOBEC3B and APOBEC3F because their exon/intron structures are similar to that of APOBEC3G, with the presence of either all eight exons or exons 1–7 (21). Other APOBEC3 genes of humans have at most exons 1–5 (21). Inclusion of these genes would render the phylogenetic analysis unreliable because of the reduced number of sites that can be used for tree-making. Mice have only one APOBEC3 gene, and it is located in the chromosomal region that is syntenic to human chromosomal 22q13, where the human APOBEC3 genes are found (22). The phylogeny (Figure 2) shows that the seven primate APOBEC3G sequences form a statistically supported monophyletic group and that their relationships are consistent with the known species phylogeny. These results indicate that the seven primate APOBEC3G sequences are indeed orthologous.

View larger version (21K): [in this window] [in a new window]   Figure 1. Alignment of APOBEC3G sequences of seven primates. Dot indicates identity to the human sequence and dash indicates a gap. Asterisk indicates position 128, which mediates species-specific interactions between APOBEC3G and vif in humans and African green monkeys. The sequences from the pygmy chimpanzee, gorilla and orangutan were determined in this study and all other sequences were retrieved from GenBank.

 

View larger version (17K): [in this window] [in a new window]   Figure 2. Phylogenetic tree of the primate APOBEC3G sequences. The tree was reconstructed by the neighbor-joining method with protein p-distances. Shown at interior nodes are bootstrap percentages derived from 2000 replications. Human APOBEC3B, human APOBEC3F and mouse APOBEC3 are used for examining the monophyly of the primate APOBEC3G sequences.

 
Positive selection on primate APOBEC3G
The APOBEC3G protein alignment reveals high sequence variation among the seven primates (Fig. 1). To examine whether this is a result of positive selection, we computed the synonymous (d_S) and non-synonymous (d_N) distances between each pair of the sequences. Higher d_N than d_S is observed in 18 of 21 pairwise comparisons (Fig. 3), suggesting the action of positive selection. Because these pairwise distances are not independent from each other, we adopted a phylogeny-based approach to statistically test the hypothesis of positive selection (23). The phylogentic relationships of the seven primates are well established, as shown in Figure 4 (same as in Fig. 2). On the basis of this tree, we inferred the ancestral APOBEC3G gene sequences at all interior nodes of the tree. Because the species involved are closely related, this inference had high reliability, with the average posterior probabilities >98.5% for each of the ancestral sequences. We then counted the numbers of synonymous (s) and non-synonymous (n) substitutions on each tree branch (Fig. 4). The sums of n and s for all branches are 171 and 45, respectively. The potential numbers of non-synonymous (N) and synonymous (S) sites are 808.9 and 301.0, respectively, as estimated by the modified Nei–Gojobori method (24). Thus, the n/s ratio (3.80) is 1.4 times the N/S ratio (2.69), and their difference is statistically significant (P=0.020, binomial test). The binomial test used here is more conservative than the Fisher's exact test used in the literature (23), and is more appropriate because multiple substitutions may have occurred at individual sites (25). Fisher's exact test would have given a P-value of 0.007. To examine if the synonymous substitution rate in APOBEC3G is normal, we computed the average number of synonymous substitutions per site between the five hominoids and two Old World monkeys. This number is 0.093, slightly higher than the corresponding number (0.079) obtained from multiple intron and non-coding sequences of primate genomes (26), revealing that the synonymous substitution rate in APOBEC3G is not reduced. Thus, our finding of a significantly higher rate of non-synonymous substitutions than that of synonymous substitutions strongly suggests positive selection in APOBEC3G.

View larger version (10K): [in this window] [in a new window]   Figure 3. Pairwise comparisons of dS and dN among seven primate APOBEC3G sequences.

 

View larger version (18K): [in this window] [in a new window]   Figure 4. Numbers of synonymous (s) and non-synonymous (n) substitutions in primate APOBEC3G. Shown above each branch is the n/s value. N and S are the potential numbers of non-synonymous and synonymous sites, respectively. Asterisk indicates species that are known to be infected by HIV/SIV and the bold-lined branches are the terminal branches for these species.

 
To investigate whether the positive selection is localized in small regions of the gene, we conducted a sliding-window analysis of the pairwise d_S and d_N among the seven primate sequences and then computed average d_S and d_N for each window. Each non-overlapping window contains 30 codons, except for the first (23 codons) and last windows (18 codons) because of the presence of gap sites. For all windows except one, higher average d_N over d_S is observed. However, the difference between the average d_N and d_S is not statistically significant in any of the windows, apparently due to the large sampling errors (Fig. 5). The N-terminal window shows the highest d_N and it is also significantly higher than the average d_N over the entire gene (P<0.05, Z-test). These results show that the positive selection on APOBEC3G is not localized to small regions and that it is particularly strong in the N-terminus.

View larger version (16K): [in this window] [in a new window]   Figure 5. Sliding-window analysis of average dS (open bars) and dN (closed bars) among the seven primate APOBEC3G sequences. Each non-overlapping window contains 30 codons, except for the first (23 codons) and last windows (18 codons) because of the alignment gaps. The thin and bold dotted lines show the average dS and dN for the entire sequences, respectively. The error bar shows one standard error.

 
No variation in d_N/d_S among lineages
As APOBEC3G is known to be antagonistic to HIV/SIV vif, one may examine if the positive selection on APOBEC3G is stronger in species infected by HIV/SIV. Of the seven primate species concerned here, the human, chimpanzee, macaque and African green monkey are infected by HIV/SIV, whereas the pygmy chimpanzee, gorilla and orangutan are not. In the tree of Figure 4, the n/s ratio for the exterior branches leading to the human, chimpanzee, macaque and African green monkey (bold lines in Fig. 4) is 47.5/11.5=4.13 and is not significantly different from the ratio (123.5/33.5=3.69) for the other branches of the tree (P>0.4, Fisher's exact test). We also used a likelihood method (27) to examine if there is variation in d_N/d_S among the branches of the tree. This computation showed that when all the branches were assumed to have the same d_N/d_S ratio, this ratio was estimated to be 1.37 and the log-likelihood was –2575.78. When the d_N/d_S ratio was allowed to vary among all the branches the log-likelihood became –2572.02 and was not significantly higher than the previous number (P>0.5, ²-test). Thus, the likelihood method also did not detect variation in the d_N/d_S ratio among branches.

Human polymorphisms
It is of interest to measure the intraspecific polymorphism of APOBEC3G and compare the polymorphism data with the interspecific divergence data, as such comparisons can reveal the process of natural selection. We sequenced exons 2–7 in 23 human individuals of different geographic ancestry (see Materials and Methods) and identified seven polymorphic sites (Table 1). Of these, four are synonymous and three are non-synonymous. Nucleotide diversity (26) is 0.0010 per site and Watterson's (26) is 0.0014 per site. Neither Tajima's test (28) (D=–0.789, P>0.2) nor Fu and Li's test (29) (D*=–1.82, P>0.05; F*=–1.75, P>0.05) rejects neutrality for the polymorphism data. Furthermore, the heterogeneity test (30) does not detect a difference in Tajima's D between synonymous and non-synonymous sites (P>0.1). These results could be due to no deviation of the polymorphic pattern from neutrality or due to the lack of statistical power as the number of polymorphic sites is small. The intraspecific n/s ratio (3/4=0.75) is significantly lower than the interspecific n/s ratio (171/45=3.8, Fig. 4) among the seven primates (P=0.043, Fisher's exact test). The excess of non-synonymous substitutions between species likely resulted from fixations of species-specific advantageous mutations (31). The McDonald–Kreitman test (31) performed earlier is traditionally used for comparing closely related species. When the divergence among the human, chimpanzee and pygmy chimpanzee is considered, the interspecific n/s ratio is similarly high (18/5=3.6). However, owing to the small number of substitutions between the human and chimpanzees, the difference between the intraspecific and interspecific n/s ratios is no longer statistically significant (P=0.096).

View this table: [in this window] [in a new window]   Table 1. DNA sequence variations in human APOBEC3G

 
Strong bias in charge changes
In many proteins, positive selection results in non-random amino acid substitutions (24,32,33). To investigate whether this is the case in APOBEC3G, we counted the numbers of conservative and radical non-synonymous substitutions on each branch of the tree (Fig. 6). Conservative non-synonymous substitutions are defined as those that do not alter the charge of the encoded amino acids whereas radical substitutions alter the charge (34). We found a total number of r=93 radical substitutions and c=78 conservative substitutions in the tree. The potential numbers of radical and conservative sites are R=322.6 and C=486.5, respectively. The radical substitution rate (r/R=0.288) is significantly greater than the conservative substitution rate (c/C=0.160) (P<0.001, binomial test). This is in contrast to the situation in most mammalian genes where the radical substitution rate is lower than the conservative rate (34). The number of synonymous substitutions per site for the entire tree is s/S=45/301=0.150 (Fig. 4). The rate of conservative non-synonymous substitutions (0.160) is similar to the rate of synonymous substitutions (0.150), whereas the rate of radical non-synonymous substitutions (0.288) is significantly higher than the synonymous rate (P<0.001, binomial test). These results strongly suggest that the positive selection favors alterations of amino acid charge in APOBEC3G evolution. We also tested the hypothesis that selection favors alterations of amino acid polarity or size and polarity (34), but obtained no supporting evidence.

View larger version (18K): [in this window] [in a new window]   Figure 6. Numbers of conservative non-synonymous (c) and radical non-synonymous (r) substitutions in primate APOBEC3G. Conservative non-synonymous substitutions do not alter the charge of the encoded amino acid, whereas radical non-synonymous substitutions do. Shown above each branch is r/c. R and C are the potential numbers of radical non-synonymous and conservative non-synonymous sites, respectively.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
What is driving the rapid pace of amino acid substitution in primate APOBEC3G? Because APOBEC3G is involved in an antagonistic interaction with the vif protein of HIV/SIV, one naturally thinks that HIV/SIV is the driving force. Consistent with this hypothesis, vif has been found to evolve rapidly by positive selection (20). However, because viral genes tend to evolve rapidly and almost all genes in the HIV genome are shown to be subject to positive selection (20), it is possible that the rapid evolution of APOBEC3G may not be due solely to the rapid evolution of vif.

Our sliding-window analysis of APOBEC3G showed that d_N exceeds d_S for virtually every window and that the d_N/d_S ratio is particularly high in the N-terminus. Unfortunately, there is no available 3D structure for APOBEC3G or any animal APOBEC protein and the interacting residues between APOBEC3G and vif are unknown. This makes it difficult to test whether there are coordinated amino acid substitutions in the two proteins and whether the selective pressure on APOBEC3G is related to vif. If HIV/SIV infection is indeed the selective agent, we expect that the d_N/d_S ratio, a measure of the intensity of positive selection, will be higher for the primate species that are infected by HIV/SIV than for those that are uninfected. However, we found no significant variation in d_N/d_S among primate lineages. This negative result may have multiple reasons. First, HIV/SIV infection of a species may be a relatively recent event and it is not easy to correctly assign a branch in the tree of Figure 4 to either ‘with HIV/SIV infection’ or ‘without infection’. Second, even when a species is infected by HIV/SIV, antagonism between APOBEC3G and vif may not be necessary if there are other mechanisms preventing viral replication in the host. Our unexpected finding, however, is not the lack of positive selection in species infected by HIV/SIV. Rather, it is the presence of positive selection in species that are not infected by HIV/SIV. For instance, the d_N/d_S ratio is 1.59 in the exterior branch leading to orangutan, an ape not known to be infected by SIV. Furthermore, despite the fact that HIV infection in humans has occurred only in the past century (35) and it should not have affected the d_N/d_S ratio for either divergence or polymorphism of human APOBEC3G, the observed d_N/d_S ratio is 2.97 in the human branch following the separation of humans from chimpanzees.

Because APOBEC3G is a cytidine deaminase and its antiviral mechanism does not depend on the recognition of specific cDNA sequences, it is possible that APOBEC3G is also involved in host defense against other viruses. In fact, recent studies showed that the antiviral effect of APOBEC3G extends to other retroviruses including murine leukemia virus and equine infectious anemia virus (6,8). Furthermore, there is a report of an excess of G-to-A mutations in the sequences of the human hepatitis-B virus (HBV) (36). HBV has a DNA genome, but there is an obligatory reverse transcription step from full-length genomic RNA to DNA within the viral capsid structure in the cytoplasm (11). It is possible that these hypermutations were induced by APOBEC3G. Indeed, a recent paper shows inhibition of HBV replication by APOBEC3G (37). Interestingly, these three viruses do not contain homologs of vif though it is possible that these viruses possess other genes that are functionally equivalent to vif. On the other hand, vif is present in virtually all lentiviruses, to which HIV/SIV belong. One can imagine that vif and APOBEC3G are under different selective pressures. vif has to evolve specific structures and sequences to recognize and bind APOBEC3G, but APOBEC3G only requires substitutions that abolish viral recognition although maintaining its cytidine deaminase activity. In other words, APOBEC3G is likely under diversifying rather than directional positive selection (25). It is possible that the conservation of a small number of important residues is sufficient to maintain the basic structural and functional requirement for cytidine deaminase activity. The interaction of APOBEC3G and vif is thus different from most interactions between host immune systems and pathogens whereby the host has to recognize the pathogen whereas the pathogen needs to escape host recognition. This feature of the APOBEC3G–vif interaction might explain why there is high d_N/d_S ratio in almost every region of APOBEC3G, as any region of APOBEC3G may be targeted by vif (or its functional equivalents in non-lentiviruses). The overall d_N/d_S ratio may be further enhanced if APOBEC3G interacts with multiple viruses in any host species.

Recently, several groups reported that position 128 of APOBEC3G (Fig. 1) is critical in mediating the species-specific interaction between APOBEC3G and vif in humans and African green monkey (38–41). That is, a single amino acid change from Asp to Lys at position 128 makes human APOBEC3G sensitive to the vif protein from African green monkey SIV but resistant to that of HIV. Conversely, the reciprocal change in African green monkey APOBEC3G renders it sensitive to the vif of HIV, but resistant to that of African green monkey SIV. Interestingly, the Asp is conserved among the five hominoids and Lys is conserved among the two Old World monkeys examined. Because Asp is negatively charged and Lys is positively charged, Schröfelbauer et al. (38) hypothesized that the APOBEC3G–vif interaction is mediated by electrostatic interaction. Our observation of positive selection for charge-altering amino acid substitutions lends strong support to this hypothesis. An interesting question is why there are so many amino acid changes in the evolution of primate APOBEC3G if one amino acid change can prevent it from being targeted by vif. This question may have two answers. First, APOBEC3B may be targeted by more than one virus, as discussed earlier, and therefore requires multiple substitutions to avoid recognition by all these viruses. Second, because HIV/SIV has a high mutation rate, one can imagine that any changes in APOBEC3G will be quickly counteracted by changes in vif. The observation of one effective amino acid change of APOBEC3G in a lab does not mean that this change is permanently effective in nature. Most likely, evolution has witnessed many rounds of such counteracting amino acid substitutions in APOBEC3G and vif. Because HIV has a much higher rate of mutation than primates do, this battle will ultimately be won by HIV/SIV unless the infectivity and lethality of the virus decrease or new antiviral mechanisms appear in primates.

Given this, it is noteworthy that the APOBEC3 gene family expanded in primates (21). This family contains only one gene (APOBEC3) in rodents, but as many as seven genes in humans (one or two of them may be pseudogenes) (21,22). The tree in Figure 2 indicates that the expansion started before the separation of hominoids from Old World monkeys, but the details of the expansion are unclear as the orthologs of most APOBEC3 genes have not been sequenced in other primates. It is currently unknown whether APOBEC3G is the only antiviral member in the family or the entire family possesses this activity. It is also unclear whether APOBEC3G performs cellular functions other than host defense against virus, as APOBEC1, a paralog of APOBEC3G, is known to have a physiological role in editing apoB mRNA. DNA editing appears an efficient mechanism of generating hypermutation and causing subsequent decay of reverse transcripts. The dramatic decline of retrotransposon activities in primates, since 35–50 million years ago (42), may be a result of the rise of the APOBEC3 family. The primate genome has been infected by many retroviruses in the past 30 million years and is still threatened by new retroviral infections (43,44). The intriguing possibility that the expansion of the APOBEC3 family in primates is a response to viral infection should be seriously considered.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
DNA amplification and sequencing
The human APOBEC3G gene has eight exons, of which exons 1 (17 nt) and 8 (15 nt) are very short. We amplified all eight exons from the genomic DNAs of a pygmy chimpanzee Pan paniscus, a gorilla Gorilla gorilla and an orangutan Pongo pygmaeus, using MasterTaq under conditions recommended by the manufacturer (Eppendorf, Hamburg, Germany). The products were then purified and sequenced from both directions using the dideoxy chain termination method with an automated sequencer. The polymerase chain reaction primer sequences are available upon request. We also amplified and sequenced exons 2–7 of 23 human individuals from different geographic origins (three Pygmy Africans, seven African Americans, five Caucasians, three Southeast Asians, two Chinese, two Pacific Islanders, one Andes Indian) to detect DNA sequence polymorphisms.

Evolutionary analysis of APOBEC3G sequences
APOBEC3G gene sequences from the human Homo sapiens (GenBank accession number: NM_021822), chimpanzee Pan troglodytes (AY331715), macaque Macaca mulatta (AY331716) and African green monkey Chlorocebus aethiops (AY331714) were obtained from the GenBank. Human APOBEC3B (NM_004900), human APOBEC3F (NM_145298) and mouse (Mus musculus) APOBEC3 (NP_084531) sequences were similarly retrieved.

The protein sequences of primate APOBEC3G, human APOBEC3B, human APOBEC3F and mouse APOBEC3 were aligned using CLUSTAL X (45). The DNA sequence alignment of the primate APOBEC3G genes was also obtained using CLUSTAL X. Gene trees were reconstructed using the neighbor-joining method (46), with 2000 bootstrap replications (47). MEGA2 (48) was used for the phylogenetic analysis. The number of synonymous nucleotide substitutions per synonymous site (d_S) and that of non-synonymous substitutions per non-synonymous site (d_N) were computed using the modified Nei–Gojobori method (24), with an estimated transition/transversion ratio of 1.7. On the basis of the phylogeny of the seven primates involved, we inferred ancestral APOBEC3G sequences at all interior nodes of the tree using the distance-based Bayesian method (49). The numbers of synonymous (s) and non-synonymous (n) substitutions on each branch of the tree were then counted. Radical and conservative non-synonymous substitutions with regard to amino acid charge, polarity, and size and polarity were computed following Zhang (34). Population genetic analyses were performed using DnaSP (50).

	ACKNOWLEDGEMENTS

 
We thank Shaneen Braswell for technical assistance and two anonymous reviewers for constructive comments. This work was supported by a startup fund from the University of Michigan and National Institutes of Health grant GM67030 to J.Z.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Department of Ecology and Evolutionary Biology, University of Michigan, 3003 Natural Science Building, 830 N. University Avenue, Ann Arbor, MI 48109, USA. Tel: +1 7347630527; Fax: +1 7347630544; Email: jianzhi{at}umich.edu

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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