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Human Molecular Genetics Advance Access originally published online on May 22, 2006
Human Molecular Genetics 2006 15(13):2106-2113; doi:10.1093/hmg/ddl134
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

TRPV6 exhibits unusual patterns of polymorphism and divergence in worldwide populations

Joshua M. Akey1,*, Willie J. Swanson1, Jennifer Madeoy1, Michael Eberle1 and Mark D. Shriver2

1 Department of Genome Sciences, University of Washington, Seattle, WA, USA and 2 Department of Anthropology, Pennsylvania State University, State College, PA, USA

* To whom correspondence should be addressed at: Department of Genome Sciences, University of Washington, 1705 NE Pacific Street, PO Box 357730, HSB J-279 Seattle, WA 98195-7730, USA. Tel:+1 2065437254; fax: +1 2066857301; Email: akeyj{at}u.washington.edu

Received April 7, 2006; Accepted May 17, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
A striking footprint of positive selection was recently identified on chromosome 7q34–35 that spans at least 115 kb and encompasses four known genes (KEL, TRPV5, TRPV6, EPHB6). The signature of selection was observed in only one of the two populations analyzed suggesting the action of geographically restricted selective pressures. However, as only two populations were analyzed, it remains unknown whether the signature of selection extends to additional populations. To address this issue and begin to dissect the evolutionary history of this region in more detail, we performed an in-depth population genetic analysis on TRPV6, which is a calcium-permeable ion channel thought to mediate the rate-limiting step of dietary calcium absorption. We demonstrate that the rate of TRPV6 protein evolution is significantly accelerated in the human lineage, but only for a haplotype defined by three non-synonymous SNPs (C157R, M378V and M681T) that are nearly fixed for the derived alleles in non-African populations. Interestingly, we found that these three non-synonymous SNPs have high posterior probabilities for being targets of positive selection and are therefore strong candidates for mediating the population-specific signatures of selection in this region. In addition, we resequenced the exons corresponding to the C157R, M378V and M681T polymorphisms in 90 geographically diverse individuals and characterized their global allele frequency distribution by genotyping them in 1064 individuals from 52 populations. These data strongly suggest that the TRPV6 haplotype defined by the derived alleles at C157R, M378V and M681T conferred a selective advantage that varied spatially, and perhaps temporally, during human history.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Advances in DNA sequencing and SNP genotyping technology are providing the necessary resources to make detailed inferences on the evolutionary and demographic forces that have shaped extant patterns of human genetic variation (1,2). In particular, there is considerable interest in identifying loci that have been targets of recent positive selection (3–12). A more thorough understanding of how, where and why positive selection has acted upon the human genome will provide important insights into the evolutionary history of our species and may have significant implications for disease-related research (13,14).

Recently, Akey et al. (5). and Stajich and Hahn (15) described a striking signature of positive selection on chromosome 7q34–35 that spans at least 115 kb and encompasses four known genes (KEL, TRPV5, TRPV6, EPHB6). Interestingly, the signature of selection was observed in only one of the two populations analyzed (European-Americans and not in African-Americans), which is consistent with the action of local adaptation. Sliding window analyses across the entire 115 kb region demonstrated that TRPV6 was located in the most significant regions of both Tajima's D and reduced nucleotide diversity (5) and is thus a strong candidate for mediating the observed signature of selection.

TRPV6 belongs to the mammalian transient receptor potential superfamily, which consists of 27 genes organized into six subfamilies (16,17). TRPV6 encodes a six-transmembrane polypeptide subunit that assembles into tetramers to form cation-permeable pores (18) and is expressed in a wide variety of tissues, but is found at the highest levels in the kidney, placenta and intestine (19). TRPV6 and TRPV5 are unique from other TRP family members, as they are the most calcium-selective and possess strong inwardly rectifying currents (17) and are thought to serve as the rate-limiting step of dietary calcium absorption (17,20,21). To begin to dissect the evolutionary history of this region in more detail, we performed an in-depth population genetic analysis on patterns of TRPV6 polymorphism and divergence in geographically diverse human populations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Rate of TRPV6 Protein evolution is significantly accelerated in the human lineage
We first investigated the patterns of TRPV6 protein evolution by comparing the number of non-synonymous substitutions per non-synonymous site (dN) with the number of synonymous substitutions per synonymous site (dS) using orthologous sequences from human, mouse, rat and chimpanzee. Under neutrality, the ratio of dN/dS is expected to be equal to 1, whereas a dN/dS ratio >1 suggests the action of adaptive protein evolution. All dN/dS calculations were performed with the CODEML program of PAML version 3.14 (22).

Initially, we tested for variation in the dN/dS ratio between lineages (23,24) by comparing a model with one dN/dS ratio for all lineages with a model where each lineage had a separate dN/dS ratio estimated from the data. This analysis revealed significant variation in the dN/dS ratio between lineages ({chi}42=21.6, P<0.001) and therefore heterogeneity in the selective forces shaping patterns of TRPV6 protein evolution across species. Specifically, in the mouse, rat and chimpanzee lineages the dN/dS ratio was 0.05, 0.05 and 0.14, respectively, which indicates significant functional constraint (Fig. 1A). However, in the human lineage, dN/dS is substantially higher (Fig. 1A), which is consistent with the action of positive selection.


Figure 1341
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  		Figure 1. TRPV6 protein evolution is accelerated in the human lineage. (A) Phylogenetic tree of TRPV6 orthologs in human, chimp, rat and mouse. Branch lengths are proportional to the indicated dN/dS ratios. The numbers in parentheses summarize the results of the test for accelerated evolution in the human lineage (see text for details), which estimates that 2.6% of sites have a dN/dS >1 (P=0.024). (B) The posterior probability of having evolved under positive selection is shown for each amino acid residue. The dashed red line indicates a posterior probability of 0.90, and the three polymorphic non-synonymous sites are shown. The schematic below summarizes the major structural features of TRPV6. ANK and TM denote ankyrin and transmembrane domains, respectively.

 
On the basis of previous analyses of human polymorphism data (5,15), we had an a priori hypothesis of adaptive evolution acting along the human lineage. To test this hypothesis, we compared a model with sites along the human lineage in one of three classes (dN/dS=1, dN/dS in the interval 0–1 or dN/dS freely estimated from the data) to a model with the additional class of sites with dN/dS fixed at 1 (25). The negative of twice the difference in log likelihoods between the models was compared to a chi-square distribution with one degree of freedom. Indeed, the rate of TRPV6 protein evolution is significantly accelerated in humans ({chi}12=5.04, P=0.024) and ~2.6% of sites are estimated to have dN/dS>1 (Fig. 1A). Although the original branch-site models suffered from high false-positive rates (26), the new implementation we have used has been shown to be more robust (27).

Given the observation that TRPV6 protein evolution is accelerated in the human lineage, we used a Bayes empirical Bayes method implemented in PAML to identify amino acid residues with high posterior probabilities of having evolved under positive selection. In total, six amino acids were identified with a posterior probability of P>0.90 (Fig. 1B). Four of these residues (amino acids 17, 55, 130 and 157) are located in the first N-terminal 250 amino acids and are either within or adjacent to a cluster of five tandemly arrayed ankyrin (ANK) repeats (Fig. 1B). ANK repeats are frequently involved in mediating protein–protein interactions, and the third ANK repeat has been shown to be critical in allowing TRPV6 monomers to assemble into a functional tetrameric ion channel (28). Of the remaining two residues with high posterior probabilities, amino acid 378 is located in the first extracellular loop between transmembranes one and two, and amino acid 681 is located in the C-terminus 10 residues upstream of a calmodulin-binding domain (Fig. 1B). Although the biological significance of these amino acids is unknown, our results suggest that they are strong candidates for future functional characterization.

Interestingly, three of the amino acids with high posterior probabilities of being subject to positive selection are polymorphic in humans (C157R, M378V, and M681T) (Fig. 1B). Akey et al. (5) previously reported large allele frequency differences at these three non-synonymous SNPs between European and African-American populations. Specifically, in European-Americans, the frequency of cytosine, methionine and methionine alleles at amino acids 157, 378 and 681, respectively, was 0.98. In contrast, in African-Americans the frequency of cytosine, methionine, and methionine alleles at amino acids 157, 378 and 681 was 0.52, 0.52 and 0.50, respectively. Therefore, we tested for variation in the dN/dS ratio between lineages as described above with the common African-American haplotype consisting of arginine, valine and threonine at amino acids 157, 378 and 681, respectively. This analysis did not indicate that dN/dS varies among lineages ({chi}12=1.32, P=0.251). The African-American lineage had a dN/dS ratio of 0.07, which is substantially lower than the estimated dN/dS of 1.0 for the non-African haplotype. This is the expected result if selection has not operated on this haplotype and is consistent with Akey et al. (5) and Stajich and Hahn (15) who found that the signature of positive selection at TRPV6 was only observed in the European-American sample. Thus, evolutionary analyses based on both intraspecific polymorphism and interspecific divergence suggest that TRPV6 has experienced geographically restricted patterns of positive selection in humans.

Patterns of TRPV6 sequence variation in geographically diverse populations
The dN/dS analysis described above combined with previous analyses of nucleotide sequence variation (5,15) is consistent with the hypothesis of positive selection promoting amino acid diversification in the human lineage, and more specifically in European-Americans. However, on the basis of these data, it is unclear whether or not the signature of selection exists in additional populations. To address this issue, we resequenced TRPV6 exons 4, 9 and 15 (which include amino acid sites 157, 378 and 681) and flanking intronic sequence in 90 individuals from six geographically diverse populations and calculated standard neutrality tests of the site frequency spectrum. Specifically, we calculated Tajima's D (29), which tests for an excess of either high- or low-frequency alleles relative to neutral expectations, and Fay and Wu's H (30), which tests for a skew towards high frequency derived alleles. The signature of positive selection includes an excess of low-frequency alleles and/or an excess of high frequency derived alleles. Statistical significance was determined with the program ms (31) by performing 104 coalescent simulations assuming a standard neutral model and no recombination.

Table 1 demonstrates that Tajima's D in non-African populations is negative, indicating an excess of low-frequency alleles relative to neutral expectations. Tajima's D is significant in both the European-American and the Han Chinese samples. Similarly, Fay and Wu's H reveals an excess of high frequency derived alleles, which is significant in the European-American, Han Chinese and Middle Eastern samples (Table 1). Of the remaining non-African populations, the Japanese, South American and South East Asian samples contain too few polymorphisms to make meaningful inferences of the site frequency spectrum. However, a closer inspection of patterns of polymorphism in these samples (Fig. 2) reveals that they are fixed for the derived alleles at amino acids 157, 378 and 681. Thus, all non-African samples studied here are consistent with the hypothesis of positive selection and are fixed, or nearly fixed, for the derived alleles at amino acids 157, 378 and 681 (Fig. 2).


Figure 1342
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  		Figure 2. Graphical representation of genotypes across the sequenced regions of TRPV6. Rows correspond to individuals and columns denote SNPs. For each SNP, blue, yellow and red boxes indicate whether the individual is homozygous for the ancestral allele, heterozygous or homozygous for the derived allele, respectively. Gray boxes indicate missing data. The position of each SNP in reference to GenBank accession no. AY225461 is shown at the top of each column. Note that these SNP indentifications correspond to those of the SeattleSNPs project. Vertical bars on the left demark different populations and a dashed black box is drawn around all individuals of African descent. Gray boxes at the bottom of the figure indicate SNPs located in exons and the corresponding exon is indicated below. For SNPs located in exons, the amino acid residue is indicated by the text inside each box and the text is color-coded according to whether the SNP results in a synonymous (black) or non-synonymous (red) substitution.

 


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  		Table 1. Neutrality test statistics

 
In contrast, African samples exhibit no clear deviations from neutral expectations and possess either slightly negative or positive values of Tajima's D (Table 1). Similar results were obtained for Fay and Wu's H, except for a marginally significant value in the North African sample (Table 1). Repeating the analysis by combining all the non-African or African samples yields a consistent signature of positive selection in the former, but not in the latter (Table 1). Furthermore, levels of nucleotide diversity are an order of magnitude lower in all non-African populations relative to African populations (Table 1). Thus, patterns of sequence variation suggest that TRPV6, defined by the haplotype carrying the derived alleles at amino acids 157, 378 and 681, was subject to selection in non-African populations, extending the observations of Akey et al. (5) and Stajich and Hahn (15).

Worldwide distribution of putatively selected alleles
To better characterize the worldwide distribution of the C157R, M378V and M681T polymorphisms, we genotyped them in the HGDP-CEPH panel, which consists of 1064 individuals from 52 populations. The allele frequency distribution for all three polymorphisms was consistent with the sequence data and displayed a striking spatial pattern. Specifically, the derived alleles are either fixed or nearly fixed in all non-African populations (Fig. 3). Among African populations, there is considerable variation in derived allele frequencies. For example, the frequency of the derived nucleotide at M681T varies from 0.083 in Namibia to 0.850 in Algeria (Fig. 3 and Supplementary Material, Table S1).


Figure 1343
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  		Figure 3. Distribution of TRPV6 non-synonymous SNPs in 52 populations. Allele frequencies for the C157R, M378V and M681T polymorphisms are denoted as pie charts. Blue and red within each pie chart indicate the frequency of the ancestral and derived allele, respectively.

 
To evaluate these qualitative observations more quantitatively, we performed an analysis of molecular variance (AMOVA) (32), which partitions total genetic variation at one or more loci into distinct sources. We first considered a hierarchical AMOVA model by defining two groups, consisting of African and non-African populations, and decomposed genetic variation at C157R, M378V and M681T into three components: (i) variation among groups, (ii) variation among populations within groups and (iii) variation within populations. Table 2 demonstrates that over half of the total genetic variation at these three loci is due to variation between groups (i.e. between African and non-African populations). Of the remaining variation, 43.7% is due to variation within populations and 3.8% is due to variation among populations within regions. All three variance components are statistically significant (P<0.0001). Thus, our data indicate considerable population genetic structure at these loci.


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  		Table 2 Summary of AMOVA results

 
In addition to the hierarchical AMOVA described above, we considered two additional models to specifically address the apportionment of variation within African and non-African populations. In these analyses, we defined a single group consisting of either African or non-African populations and partitioned genetic variation into among and within-population components. There is an appreciable amount of variation between populations within Africa (15.0%, P<0.0001). In contrast, non-African populations are very homogeneous and <1% of genetic variation at C157R, M378V and M681T is due to differences between populations (P>0.05). Thus, the global distribution of allele frequencies at C157R, M378V and M681T is consistent with the hypothesis of geographically varying selective pressures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In summary, we have found considerable heterogeneity in patterns of polymorphism and divergence at TRPV6 among human populations. Specifically, TRPV6 in non-African populations is characterized by an accelerated rate of protein evolution, an excess of high frequency derived alleles, a skew in the site frequency spectrum towards low frequency alleles, low levels of nucleotide diversity and significant levels of population structure between African and non-African populations. These observations are consistent with the hypothesis that the TRPV6 haplotype defined by the derived alleles at amino acids 157, 378 and 681 was subject to selection in non-African populations.

The evolutionary history of TRPV6 in African populations, however, is less clear. Specifically, the frequency of C157R, M378V and M681T varies considerably among African populations, and the derived alleles of these polymorphisms reach a frequency of 0.85 in the North African Mozabite population (Fig. 3 and Supplementary Material, Table S1). Furthermore, although patterns of DNA sequence variation are generally consistent with neutrality, Tajima's D is 1.59 and 1.56 in individuals of South African and African-American ancestry, respectively (Table 1). Although not statistically significant, they do raise the possibility that TRPV6 may have been subject to some form of balancing selection in Africa. Consistent with this hypothesis, we analyzed the SeattleSNPs data that have sequenced 277 genes (including TRPV6) in 24 African-American and 23 European-American individuals and found that in the African-American sample only eight of the 277 genes had a Tajima's D greater than that of TRPV6 (empirical P=0.029), and none of the 277 genes had a difference in Tajima's D between African and European-Americans as large as that observed for TRPV6. Our resequencing panel and the HGDP-CEPH individuals are not well represented in Northeast African populations, which may be particularly informative to study, as they are thought to be the source of the out of Africa migrations (33). Thus, a more comprehensive analysis of African populations is necessary to make more definitive inferences about the evolutionary forces shaping patterns of TRPV6 sequence variation in Africa.

A neutral explanation for these observations seems unlikely. In theory, these data could be the result of a founder effect associated with the out of Africa dispersal, which could potentially account for the significant skew towards low-frequency alleles as assessed by Tajima's D and low levels of nucleotide diversity. A founding event may also explain the observed accelerated rate of TRPV6 protein evolution by relaxation of functional constraint. However, relaxed functional constraint would not be expected to result in a subset of sites with dN/dS statistically greater than 1 as seen for TRPV6. Furthermore, the significant excess of high frequency derived alleles is more robust to demographic perturbations such as founding effects and bottlenecks (30). In addition, the three amino acid residues that have high posterior probabilities for being targets of selection (C157R, M378V and M681T) exhibit strong levels of population genetic structure between African and non-African populations, which parallel the results of Tajima's D and Fay and Wu's H. Thus, when collectively interpreted, the most parsimonious explanation for our data is that TRPV6 was the target of positive selection whose strength varied spatially, and perhaps temporally, during human history.

The historical forces mediating the selective advantage of the TRPV6 haplotype defined by the derived alleles at amino acids 157, 378 and 681 remain speculative. Given the role of TRPV6 in dietary calcium absorption, Akey et al. (5) hypothesized that patterns of TRPV6 sequence variation may have been influenced by the agricultural revolution and more specifically the domestication of milk-producing animals, which began ~10 000 years ago. However, our current data indicate that the signature of selection at TRPV6 is shared among all non-African populations, suggesting that positive selection drove the derived haplotype defined by the C157R, M378V and M681T polymorphisms to near fixation subsequent to or coincident with the major human migrations out of Africa ~60 000–100 000 years ago (34).

Although dietary pressures unrelated to the agricultural revolution remain a plausible selective agent, alternative hypotheses should also be considered. For example, calcium is integral to proper immune function and TRPV6 has been shown to play a role in T-cell activation (35), raising the possibility that the signature of selection observed at TRPV6 is related to pathogens. Ultimately, functional studies need to be performed to determine how the C157R, M378V and M681T polymorphisms affect TRPV6 function, which may allow more biologically informed hypotheses on the potential selective agent to be developed.

Finally, it is important to note that our current data does not rule out the possibility that additional genes in this region have also been subject to selection. Indeed, using human–chimp–mouse orthologs, Clark et al. (36) found that the rate of EPHB6 (and TRPV6) protein evolution is significantly accelerated in the human lineage. EPHB6 is located ~15 kb centromeric of TRPV6 and possesses patterns of sequence variation consistent with the hypothesis of positive selection (5). However, the complete coding region of EPHB6 was sequenced in 24 African-Americans and 23 European-Americans, and no non-synonymous or synonymous SNPs were found with large allele frequency differences between populations (5). Therefore, although protein-coding regions of EPHB6 may have experienced positive selection in the human lineage, it is unlikely to account for the signature of local adaptation observed at TRPV6. Nonetheless, it is plausible that multiple advantageous alleles contribute to the signature of selection in this region. Adaptive processes in natural populations undoubtedly violate the assumptions of simple theoretical models of positive selection. Clearly, additional theoretical and empirical studies are needed to better understand how linked advantageous alleles, whose selective advantages vary both spatially and temporally, population demographic history and local rates of mutation and recombination synergistically interact to shape patterns of polymorphism and divergence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Samples
DNA samples used for resequencing were obtained from Coriell Cell Repositories (Camden, NJ, USA). In total, 90 individuals were sequenced from six populations. Coriell repository numbers (http://coriell.undmj.edu//) for these samples are as follows: Han Chinese of L.A. (NA17733–47, NA17749, NA17752–57, NA17759, NA17761), Japan (NA17051–60), Middle East (NA17041–50), North Africa (NA17378–84), Pygmy (NA10469–73, NA10492–96), South Africa (NA17319, NA17341–48), South America (NA17301–10) and South East Asia (NA17081–90). SNP genotyping was performed on the Human Genome Diversity Panel-Centre d'Etude du Polymorphisme Humain (http://www.cephb.fr/HGDP-CEPH-Panel/). Orthologous sequences from mouse (NM_022413 [GenBank] ) and rat (NM_053686 [GenBank] ) were obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) and the chimpanzee ortholog (ENSPTRT00000046671) was obtained from Ensembl (http://www.ensembl.org/Pan_troglodytes/index.html). Sequence data from 23 European-Americans and 24 African-Americans were obtained from the SeattleSNPs project (http://pga.gs.washington.edu/).

DNA sequencing
We used the TRPV6 VariantSEQr kit (Applied Biosystems, Foster City, CA, USA) for sequencing, which contains validated sets of PCR primers spanning all TRPV6 exons. Primer sets corresponding to exons 4, 9 and 15 were used in this study. Resequencing was performed according to manufacturer's protocol by standard PCR-based automated sequencing using Applied Biosystem's Big Dye sequencing protocol on an ABI 3130xl and ABI 3100. The sequence data for each gene were assembled onto the TRPV6 reference genomic sequence using Phred/Phrap (37,38), and the alignments were inspected for accuracy with Consed (39). Polymorphisms were identified with PolyPhred 4.0 (40). All polymorphic sites were manually verified and confirmed by sequencing the opposite strand.

SNP genotyping
SNP genotyping was performed using the ABI Prism SnapShot Multiplex system. Briefly, SNPs were amplified by PCR, PCR products pooled across loci and purified and multiplex single base extension (SBE) reactions were performed. The reaction products were resolved on an ABI 3100 capillary sequencer and genotypes automatically called with ABI's Genotyper v3.7 software. PCR and SBE primers are available upon request.

Statistical analysis
All dN/dS calculations used the CODEML program of PAML version 3.14 (22). We tested for variation in the dN/dS ratio between lineages (23,24) by comparing a model with one dN/dS ratio for all lineages compared with a model where each lineage had a separate dN/dS ratio estimated from the data. The negative of twice the difference between the nested models was compared with chi-square distribution with four degrees of freedom. To test whether adaptive evolution has promoted amino acid diversification along the human lineage, we used branch-site models (25). We compared a model with sites along the human lineage in one of three classes: dN/dS=1, dN/dS in the interval 0–1 or dN/dS freely estimated from the data to a model with the additional class of sites with dN/dS fixed at 1. The negative of twice the difference between the models was compared with a chi-square distribution with one degree of freedom. This is conservative as we should be comparing to a distribution of 50:50 mixture of chi-square and point mass at 0. To check for convergence, we ran all the analyses from three different initial values of dN/dS (0.3, 1, 3).

Standard neutrality tests of the site frequency spectrum were calculated including Tajima's D (29) and Fay and Wu's H-statistic (30). The statistical significance of these statistics was determined by comparing the observed values to 104 coalescent simulations (31), conditional on the observed sample size and number of segregating sites, assuming a standard neutral model with no recombination. Coalescent simulations were performed using the program ms [obtained from R. Hudson's website (http://home.uchicago.edu/~rhudson1/source.html)]. AMOVA was performed with Arlequin 2000 (41).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Dayna Akey for critical reading of the manuscript and providing valuable comments. J.M.A. is supported in part by a Pilot and Feasibility Award from the Clinical Nutrition Research Unit at the University of Washington and the NSF (DEB-0512279).

Conflict of Interest statement. The authors have had no involvements that might raise the question of bias in the work reported or in the conclusions, implications or opinions stated.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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