Update

Genome Analysis Neutral evolution on mammalian surfaces

Gavin C. Conant1,2

1 Division of Animal Sciences, University of Missouri, Columbia MO, U.S.A. 2 Informatics Institute, University of Missouri, Columbia MO, U.S.A.

Because of their low effective population sizes, natural Mammals and yeasts show similar ratios of surface to selection is expected to have reduced effectiveness in interior constraint organisms such as mammals. By comparing the amino In yeasts, we have recently compared the ratio of nonsynon- acid substitution rates between mammalian protein ymous to synonymous substitutions (Ka/Ks or v) between surfaces and interiors, it was found that almost a third surface and interior residues (vs and vi, respectively), find- of the surveyed failed to reject the null hypoth- ing not only that vs > vi (i.e. interior residues are more esis of neutral substitutions among surface residues. constrained) but, more surprisingly, that the ratio of vi/vs is Proteins with such partly neutral evolution nonetheless quite similar across a range of proteins [12]. Here I extend have no fewer protein interactions than do other this analysis to eight mammals (, chimpanzee, the proteins. I suggest that natural selection can function rhesus macaque, mouse, rat, cow, horse and dog). The to preserve protein interactions without requiring strict approach differs from previous work [4,7,8] in that selective conservation of the individual residue contacts that constraints are estimated for individual using impart those interactions. orthologous genes from several species, rather than by performing correlation analyses between exposure and pairwise sequence divergence. Although this technique Molecular evolution, population size and protein results in smaller samples, it allows us to understand structure the variation between genes in how structure affects The birth of the field of molecular evolution was attended selection. by a certain parental tension between the then-current It was hypothesized that the weakening of selection due state of evolutionary theory and the new ability to to mammals’ small Ne would reduce the difference in study the genetic structures of populations. This neutr- constraint between the surface and interior. Instead, Ne alist–selectionist debate continues to stimulate discus- appeared to manifest itself as an increased propensity sion as to whether the majority of the fixed sequence toward neutral evolution at the surfaces of mammalian differences between species have arisen through genetic proteins. (Here ‘neutral evolution’ is used as shorthand for drift [1–3]. the fixation of nearly neutral alleles by genetic drift.) One avenue for exploring this question is to compare Using 194 human proteins with known crystal struc- situations where the propensity for neutral variation var- tures [13], the corresponding genes were aligned with their ies. One such comparison is between the residues of a orthologs in the seven other genomes and the relative protein exposed to the solvent versus those buried in the surface exposure x of each residue was calculated (see interior of the folded structure. It is well known that the supplementary data online). The resulting alignments interior residues are subject to stronger constraints were then analyzed using two models of codon evolution. against substitution than are exposed residues [4–6]. This The first model employed was that of Muse and Gaut/ pattern is maintained over wide taxonomic ranges, in- Goldman and Yang (MG/GY) [14,15] where the ratio of non- cluding mammals [7], yeasts [8] and bacteria [5], despite synonymous to synonymous substitutions (i.e. selective the variation in effective population sizes (Ne) among these constraint) is given by the parameter vg. This model organisms that might be thought to produce differing se- was extended by allowing residues to experience a con- lective regimes. tinuous mix of selective constraints depending on their Such differences in the actions of natural selection are proportional surface exposure x (see the supplementary suggested by the nearly neutral theory of molecular evol- data online). Thus, the likelihood L of a particular site s is ution [9] that relates such selection to Ne. This theory given by: points out that, because selection is less effective in prun- x Lðsj Þþð1-xÞLðsj Þ (1) ing disadvantageous variation from small populations, s i one should observe an association between Ne and A likelihood ratio test (LRT, see the supplementary data measures of genetic diversity [10]. The implied increased online) [16] was then used to explore whether there was a propensity for neutral substitutions is potentially import- significant difference in the selective constraint on surface ant for a number of reasons: not least because neutral and interior residues. Of the alignments considered, 151 changes can profoundly affect the trajectories of biological showed significant improvement under the MGstruct model evolution [1,11]. (LRTMG-struct; Table 1) with a false-discovery rate (FDR) threshold of 0.05 [17]. The constraint ratio vi/vs is similar across these 151 Corresponding author: Conant, G.C. ([email protected]). proteins (Figure 1b) and does not differ significantly from 377 Update Trends in Genetics Vol.25 No.9

Table 1. Likelihood ratio tests (LRT) performed LRT Null Null model Alternative Alt. model ndfb No. of Groups model amino acid model amino acid significant differd substitution substitution results c parametersa parametersa

LRTMG-struct MG vg MGstruct vs, vi 194 1 151* LRTMG-struct-neu MGstruct vs, vi MGstruct/neu vs=1.0, vi 151 1 104** s s i i LRTSG-struct SG Iw,Ib SGstruct I w,Ib,Iw,Ib 151 2 150* s s i i s s i i LRTSG-struct-neu SGstruct I w,Ib,Iw,Ib SGstruct/neu I w=Ib;Iw,Ib 1 0.001 Neue 47 16** Self 104 65** s s i i s s i i -10 LRTSG-Iw-neu SGstruct I w,Ib,Iw,Ib SGIw-neu I w=1.0; I b, I w,Ib 1 <10 Neue 47 2** Self 104 77** s s i i s s i i -10 LRTSG-Ib-neu SGstruct I w,Ib,Iw,Ib SGIb-neu I b=1.0; I w, I w,Ib 1 <10 Neue 47 16** Self 104 96** aModel parameters describing the rate of nonsynonymous substitutions and their constraints. For the MG and SG models, no structural information was included: each model has only a single set of nonsynonymous parameters (vg and Iw,Ib, respectively). For the remaining models, the surface residues and interior residues were allowed to take on s s i i distinct nonsynonymous rates (vs and vi for the MG-derived models and I w,Ib,Iw and I b for the SG-derived models). bThe degrees of freedom (df) used for calculating the significance of the LRT under a chi-square distribution. For a given test, df is determined from the number of excess parameters in the more complex model. Thus, for the comparison of the MG and MGstruct models, df=1 because the MGstruct model has one extra parameter (vi and vs as opposed to simply vg). cSignificance of results from LRT: * False discovery rate 0.05; ** P0.05. dHypothesis test that the Neu and Sel sets have equal proportions of alignments that reject the null hypothesis for a given LRT (chi-square test). e Alignments that do not reject the null hypothesis of MGstruct for LRTMG-struct-neu. f Alignments that reject the null hypothesis of MGstruct for LRTMG-struct-neu. that seen in yeasts (Wilcoxon rank test, P = 0.42, One might suspect that Neu consists of cases where the Figure 1a,b). This observation reinforces our previous power to detect differences in vs is low. To examine this speculation that the limited variation in vi/vs results from issue, sequence data were first simulated under models substitutions in the protein interior being more likely to where vs 6¼ 1.0 and the power of the LRT to reject vs = 1.0 hinder folding [18]. Note however that both vi and vs are using these data was measured (see supplementary significantly larger in mammals (Wilcoxon rank test, material online). As can be seen in Figure 1d, power is P<0.002). Moreover, contrary to the situation in yeast, low for values of vs close to 1.0 but higher in other there is a significant association between vg and vi/vs parameter ranges. To study the degree of statistical sup- -10 (Spearman’s s = 0.54, P<10 ). port for the value of vs for each in Neu, I simulated data under the ML parameter estimates and determined Neutral evolution at mammalian protein surfaces whether each simulated dataset was able to reject two null Fourteen of these 151 alignments had estimates of vs >1.0. hypotheses: vs = 1.0 and vs = 0.5. The second hypothesis is Under the MG/GY model, v >1.0 indicates that the selec- included as a test of whether a sequence alignment has an tion operating on nonsynonymous substitutions is no overall strong signal of the value of vs even if that align- greater than that (potentially) operating on synonymous ment cannot reject vs = 1.0. Thus, in cases where vs is near substitutions, whereas v >1.0 is indicative of directional/ unity, the power to reject vs = 1.0 can be low, but vs might diversifying selection. To test the extent to which (nearly) still be clearly distinguishable from 0.5. The choice of vs = neutral evolution might be occurring in these genes, the 0.5 is of course somewhat arbitrary but represents a MGstruct/neu model was introduced, requiring vs = 1.0. For natural and moderate level of selection. I next defined a 47/151 (31%) of proteins (hereafter Neu), the null hypoth- measure of support q for the value of vs, given by the esis of neutral evolution among the surface residues could proportion of simulated datasets that rejected either vs = not be rejected (P>0.05; LRTMG-struct-neu; Table 1). Notably, 0.5 or vs = 1.0 at P=0.05 for a given gene in Neu. Thus, q can only 1/61 (2%) of yeast proteins fail to reject neutrality be thought of as how often an alignment like the one used (P>0.05, LRT, Figure 1b), suggesting that yeasts, with would be able to distinguish between vs = 0.5 and vs = 1.0 their larger Ne, more efficiently remove mildly deleterious with statistical significance. The average value of q across surface substitutions. Of the remaining 104 mammalian the 47 genes is 0.54 and all but three genes have q > 0.25. alignments (hereafter Sel), three have vs greater than 1.0 Thus, while it is very difficult to determine the number of (FDR-corrected P value 0.061), an observation true-positive neutral proteins, one can at least say with suggesting directional selection. The human genes in ques- some confidence that the test would have the power to tion are: OSM encoding oncostatin M, CD8A encoding the distinguish between neutrality and vs = 0.5 with relatively immune system protein CD8a, and CLEC1B encoding a high statistical confidence (P = 0.05) for approximately half member of the protein family which contains C-type lectin of the 47 genes considered. domains. Oncostatin M is a cytokine involved in controlling A second model of sequence evolution was also cell differentiation [19], whereas CD8a contributes to the employed, the similarity groups (SG) model, to see if the binding between the MHC and T-cell receptor [20]. The patterns of the amino acid substitutions support neutral- CLEC1B gene product has a less well-defined function in ity. This model categorizes amino acid substitutions the immune system [21]. according to charge [12,22], allowing one substitution rate

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in fit with the SGstruct model (LRTSG-struct; Table 1): the remaining case shows marginal significance (FDR-corrected P = 0.057). The SGstruct model was therefore compared to the s s neutral SGstruct/neu model (I w =Ib). In the Neu set, 66% of the genes do not reject SGstruct/neu (LRTSG-struct-neu; Table 1): significantly fewer of the genes in Sel (38%) fail to do so (P = 0.001, chi-square test). The Iw and Ib parameters in the SG model give the ratio of nonsynonymous charge-preserving substitutions to synonymous substitutions, and of nonsynonymous charge-altering substitutions to synonymous substi- tutions, respectively. In other words, they are akin to v in the MG/GY model, and one can test for neutrality by s s requiring I w or I b = 1.0. For both parameters, significantly more cases are found in Sel where neutrality cannot be rejected than in Sel (LRTSG-Iw-neu and LRTSG-Ib-neu; Table 1). Of the 16 genes in Neu that reject the SGstruct/ neu model, 15 genes fail to reject the null hypothesis of s (partial) neutrality for I w and four genes fail to reject s s neutrality for either I w or I b. Examples where Iw or Ib are significantly greater than 1.0 are indicators of directional selection. Of these 151 genes, only two of the three genes identified previously as indicative of directional selection were consistent with s such selection under this test: OSM (I b > 1.0; FDR-cor- s rected P=0.007) and CD8A (I w > 1.0; FDR-corrected P = 0.001).

Neutral substitutions and the protein interaction network Although surface residues are less implicated in protein folding, they still mediate protein interactions, making this apparent neutrality somewhat surprising. This is especi- ally the case as coevolution between interacting residues has been documented (reviewed in Ref. [23]). Of course, if the Neu set had been drawn from proteins with few inter- Figure 1. Yeast and mammalian proteins show similar patterns in their ratios of actions, that might explain the low surface constraint. surface to interior selective constraint but differences in the propensity for However, there is little difference between the two sets neutrality. (a) The x-axis gives the ratio of the protein interior average selective constraint to the protein surface average constraint (vi/vs); the y-axis gives twice in the proportion of genes with protein interactions: 47% the difference in ln-likelihood between a model where vs = 1.0 and a model where for Neu verses 42% for Sel (see the supplementary material this value is free to vary (LRTMG-struct-neu; Table 1). Points from yeast are shown in online). The average number of interactions per gene is blue and those from mammals in black. The dashed line gives the value of LRTMG- struct-neu (i.e. 2DlnL) equivalent to P = 0.05 (thus points below this line do not reject also similar (4.7 and 4.6, respectively; see the supple- neutrality at this significance threshold). (b) Histogram of the distribution of vi/vs mentary data online). Neither of these figures differs for the two groups (x-axis in a) with the same shading scheme. (c) Histogram of the significantly between the sets (P=0.61 and P=0.48, distribution the value of LRTMG-struct-neu for the two groups (y-axis in a), again shaded. (d) Simulations of sequence evolution illustrating the variation in power of chi-square test and Wilcoxon rank test, respectively). an LRT to reject the hypothesis of vs = 1.0 (e.g. neutrality) for different assumed There is also no difference in the proportion of proteins values of vs (x-axis) and sequence length (y-axis). The degree of shading indicates in the two sets annotated with the GO term ‘protein the proportion of simulations rejecting vs = 1.0 at P = 0.05. With a simulated value of vs = 0.9 this proportion ranges from 0.04 to 0.14, while when vs = 0.5 that binding’ [24] (P>0.5, Fisher’s exact test). Of course, it proportion ranges from 0.34 to 0.61. White points indicate the vs values and remains possible that a few interacting surface residues alignment lengthes for the neutrally evolving genes discussed in the text. are under selective constraint but that the model used here is insufficiently sensitive to detect them. between residues with the same charge (Iw) and a second (Ib) between those of differing charge. Under neutrality, Implications of surface neutrality for molecular substitutions altering the charge should be no less prob- evolution able than charge-preserving substitutions. The full model Of the proteins surveyed, at least 21% show neutral evol- (SGstruct) employed has independent values of Iw and Ib for ution among surface residues (the 31 gene products that i s i s the surface and interior residues (I w,Iw and I b,Ib). The fail to reject either MGstruct/neu or SGstruct/neu). Note how- s s signature of neutral evolution is thus I w =Ib. ever, that relatively few residues are completely exposed: The SG model distinguishes surface and interior resi- on average only 7% of a protein has solvent exposure of 75% dues: all but one of the 151 alignments where LRTMG-struct or more. Although these results might appear at variance is significantly large also show a significant improvement with a previous analysis that rejected neutrality [7], the

379 Update Trends in Genetics Vol.25 No.9 difference is likely to be due to the fact that the former accompanied by any necessary change in the function of study aggregated data across genes. the network. My simulations suggest that low statistical power alone does not account for all of the observed neutrality. This Acknowledgements contention is supported by the fact that all 151 alignments I would like to thank M. Woolfit and four anonymous reviewers for critical ˚ have sufficient evolutionary signal to distinguish between comments on the manuscript and A.Pe´rez-Bercoff, D. Gonzalez Knowles and A. McLysaght for helpful discussions regarding ortholog inference. surface and interior residues (vs and vi are statistically This work was supported by the Reproductive Biology Group of the Food distinct). On a related note, Berglund et al. [25] recently for the 21st Century program at the University of Missouri. suggested that some inferred instances of directional selec- tion might be better explained by mutational biases. There Supplementary data are three reasons for thinking it unlikely that similar Supplementary data associated with this article can be forces are at work here. First, the approach used here found at doi:10.1016/j.tig.2009.07.004. Alignments, surface identifies residue exposure independently of and prior to exposures and tree files for the 194 alignments analyzed are constraint, rather than detecting positive selection directly provided on the author’s website: (http://web.missouri.edu/ from sequences. Second, no association was found between conantg/data/mammal_struct_evol/alignments_trees_ base composition and neutrality (see the supplementary rates.tar.gz). material online). 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Genome Analysis Group I-intron trans-splicing and mRNA editing in the mitochondria of placozoan animals

Gertraud Burger, Yifei Yan, Pasha Javadi and B. Franz Lang

Robert Cedergren Centre for Bioinformatics and Genomics, De´ partement de Biochimie, Universite´ de Montre´ al, 2900 Boulevard Edouard-Montpetit, C.P. 6128, Montre´ al, Que´ bec, H3T 1J4, Canada

Placozoa – the simplest known free-living animals – either within or close to Cnidaria (e.g. Ref. [6])orasa have been considered primitive, early diverging metazo- sister clade to Porifera (sponges) / Ctenophora (comb ans based on mitochondrial genome structure and phy- jellies) [7]. Multi-protein phylogenies are similarly incon- logeny. Here we reanalyze placozoan mitochondrial clusive. A report on the Trichoplax nuclear genome [8] DNAs, reported to include a highly unorthodox, presents a phylogeny in which Placozoa emerge before fragmented and incomplete cox1 gene. We discover Cnidaria plus Bilateria, but statistical tests of alternative overlooked exons and split group I introns that mediate tree topologies leave the position of Trichoplax as unsup- trans-splicing of the discontinuous placozoan cox1. ported, and taxon sampling is limited (only one demos- Furthermore, we find that cox1 expression involves ponge, one placozoan, and two cnidarians; there are no U-to-C editing, reconstituting an otherwise invariant, representatives of other sponge lineages or ctenophores). essential histidine involved in copper binding. These Independent analysis using nuclear plus mitochondrial atypical features qualify placozoan mitochondrial gene sequences, but with similarly limited species sampling and genome organization as derived rather than primi- (for example, outgroup species close to Metazoa, such as tive. Whether the Placozoa diverged early or late during Ministeria and Capsaspora, were not included) provides metazoan evolution remains unresolved by mitochon- support for monophyly of Placozoa plus non-bilaterian drial phylogeny animals [9], but this is probably a long-branch attraction (LBA) artifact. Finally, a phylogenomic analysis with rich taxon sampling and the more reliable CAT model Are Placozoa primitive or derived animals? recovers sponge monophyly and certain deep animal Trichoplax adhaerens, an organism thriving in tropical relationships, yet fails to position the placozoan repre- waters, is the simplest known free-living animal [1].It sentative in the tree with confidence (62% bootstrap grows as a flat disk, 0.5–3.0 mm wide and 25 mm thick, support [10]). has only four different somatic cell types, and lacks organs, By contrast, two phylogenies constructed with concate- differentiated tissues, and body symmetry [2]. Tradition- nated mitochondrion-encoded proteins strongly indicate ally, T. adhaerens has been the only recognized species of that the Placozoa diverged later than the Bilateria, a sister the phylum Placozoa, but recent rRNA and ITS genotyping group to the Cnidaria and sponges (in other words, Placo- studies distinguish five different ‘highly divergent’ pla- zoa plus non-bilaterian Metazoa are a sister group to cozoan clades. The simple placozoan body plan has been Bilateria) [5,11]. Neverthless, as in the examples discussed interpreted by some authors as genuinely primitive (pla- above, Placozoa might be misplaced owing to poor taxon cing Placozoa as the deepest divergence within Metazoa), sampling (only three Porifera and four to five Cnidaria), but by others as reduced-derived (see Refs. [2–5] and and potential LBA might artifactually draw Placozoa references therein). towards the distant, fast-evolving fungal outgroup and/ The phylum’s phylogenetic position inferred from mol- or the extremely fast-evolving Bilateria. Indeed, the same ecular data has been equally controversial (Figure S1). set of mitochondrion-encoded proteins, but with broader With only marginal statistical support, nuclear rRNA taxon sampling, fails to provide significant support for data with broad metazoan taxon sampling place Placozoa placozoan divergence, and even yields different tree topol- ogies with different evolutionary models [12]. The recent Corresponding author: Lang, B.F. ([email protected]). availability of numerous complete mitochondrial genomes

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