A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pairwise Zscore probabilities
 Olivier Bastien^{1, 2},
 Philippe Ortet^{3},
 Sylvaine Roy^{4} and
 Eric Maréchal^{1}Email author
DOI: 10.1186/14712105649
© Bastien et al; licensee BioMed Central Ltd. 2005
Received: 25 November 2004
Accepted: 10 March 2005
Published: 10 March 2005
Abstract
Background
Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pairwise sequence alignments, respect probabilistic properties of Zscores (Monte Carlo methods applied to pairwise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction.
Results
We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pairwise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Zscore probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Zscores. Deduced trees, called TULIP trees, are consistent with multiplealignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny.
Conclusion
The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pairwise genome comparisons based on Zscore computations.
Background
Past events that gave birth to biological entities can be tentatively reconstructed based on collections of descriptors traced in ancient or presentday creatures. Using genomic sequences, an estimate of the relative time separating branching events, previously supported by geological records, could be formalized using mathematical models. The use of proteins for evolutionary reconstructions was vastly explored as soon as the first amino acid sequences were made available [1–9]. The rich biological information contained in protein sequences stems from their being, on the one hand, translation of genes that reflect the history of genetic events to which the species has been subjected, and on the other hand, effectors of the functions constituting a living creature [10] Since protein sequences are encoded in a 20amino acid alphabet, they are also considered to embody more informationpersite than DNA or RNA [11]; they also exhibit smaller compositional trends [12, 13]. When compared, sequences that share substantial features are considered as possible homologues [14], based on the fundamental postulate that can be simply stated as "the closer in the evolution, the more alike and conversely, the more alike, probably the closer in the evolution".
As summarized by Otu and Sayood [15], the techniques of molecular phylogenetic analyses can be divided into two groups. In the first case, a matrix representing the distance between each pair of sequences is calculated and then transformed into a tree. In the second case, a tree is found that can best explain the observed sequences under evolutionary assumptions, after evaluation of the fitness of different topologies. Some of the approaches in the first category utilize distance measures [16–19] with different models of nucleotide substitution or amino acid replacement. The second category can further be divided into two groups based on the optimality criterion used in tree evaluation: parsimony [20, 21] and maximum likelihood methods [22, 23]. For a detailed comparison of these methods see [24].
In phylogeny inference based on distance methods, features separating related proteins are used to estimate an observed distance, also called the pdistance, the simplest measure of which is just the number of different sites between proteins. Divergence time (t), also called genetic distance or evolutionary time, is calculated from the pdistance, depending on assumptions derived from evolutionary models [11, 24]. For example, the assumption that mutational events happen with equal probability at each site of any sequence leads to the molecular clock model [2]. Although widely used, it is wellknown to be unrealistic and numerous corrections have been proposed to refine it [19, 25, 26]. By definition, the distance matrix is given as T = (t_{ ab }) where a and b represent the homologous sequences from the analyzed dataset. Tree reconstruction algorithms are then applied to these matrices [11, 24]. Eventually, phylogenetic trees corresponding to the classified sequences are statistically evaluated with bootstrap methods and, when available, calibrated using dated fossils [25, 26].
Doolittle et al. [27] have proposed methods for converting amino acid alignment scores into measures of evolutionary time. Similarity between amino acids [28–30] provides a way to weight and score alignments [31]. In practice the optimal alignment of two sequences (a and b) is determined from the optimal score s(a,b) [25, 27], computed with a dynamic programming procedure [32, 33]. In aligned sequences, conservation is measured at identical sites, whereas variation is scaled at substituted sites. To estimate the variation/conservation balance, the pdistance can be given as a function of f_{ id }, the fraction of identical residues: pdistance = 1  f_{ id }. To take into account that multiple mutations can happen at the same site, an expression of f_{ id }was proposed by Doolittle et al. [27] using s(a*,b*), the score obtained from randomized a and b sequences [34] and s_{ id }, the average score of the sequences compared with themselves [19, 25, 27]:
To connect pairwise alignments and phylogeny, divergence time has been approximated:
t(a,b) = λ log[f_{ id }(a,b)] (2)
introducing a Poisson correction [2] as a reasonable stochastic law relating amino acid changes and elapsed time. As mentioned earlier, adjustments and corrections of equation (2) were proposed to fit more realistically the complexity of evolution [11, 25, 35]. This attempt of unification helped reconstructing phylogeny of major lineages [27]. However, detailed phylogenic trees obtained from evolutionary close sequences are not satisfactory. In practice, phylogenies are reconstructed based on multiple alignments. Multiple alignment based (MAB) trees are recalculated when incremented with additional sequences; although MAB methods are usually considered accurate, numerous cases of inconsistencies (incongruence) between observed data and deduced MAB trees are recorded (see [15, 36]).
Here, we reexamine the estimate of the pdistance between two homologous sequences, based on f_{ id }, as a source for geometric positioning, divergence time calculations and evolutionary reconstruction. We based our model on mathematical properties that alignment scores should respect; i) information theory [37, 38] applied to sequence similarity, ii) algorithmic theory applied to alignment optimization [28] and iii) alignment probability, particularly in conformity with the TULIP theorem [39]. We used these properties as a framework of constraints to build a geometric representation of a space of probably homologous proteins and define a theoretically explicit measure of protein proximity. This unified model conserves information in the way physical models conserve matter or energy. The obtained representation of protein sequences is unaltered by adding or removing sequences. Applications include therefore the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences.
Results and discussion
Pairwise sequence alignment scores in information theory
Criteria to measure the variation/conservation balance between proteins should embody as much as possible the structural and functional potentiality within sequences of amino acids. In the absence of explicit physical criteria, amino acid similarity was solved empirically by measuring amino acid substitution frequencies in alignments of homologous sequences [30, 40]. Given two amino acids i and j, the similarity function s(i,j) was set as:
Proposition 1
Amino acid substitution matrix values are estimates of the mutual information between amino acids in the sense of Hartley [37, 38]. Consequently, the optimal alignment score computed between two biological sequences is an estimate of the optimal mutual information between these sequences.
Proof
Given a probability law P that characterizes a random variable, the Hartley selfinformation h is defined as the amount of information one gains when an event i occurred, or equivalently the amount of uncertainty one loses after learning that i happened:
h(i) = log(P(i)) (4)
The less likely an event i, the more we learn about the system when i happens. The mutual information I between two events, is the reduction of the uncertainty of one event i due to the knowledge of the other j:
I_{j→i}= h(i)  h(i/j) (5)
Mutual information is symmetrical, i.e. I_{j→i}= I_{i→j}, and in the following will be expressed by I(i;j). The self and mutual information of two events i and j are related:
h(i ∩ j) = h(i) + h(j)  I(i;j) (6)
If the occurrence of one of the two events makes the second impossible, then the mutual information is equal to  ∞. If the two events are fully independent, mutual information is null. The empirical measure of the similarity between two amino acids described in equation (3) can therefore be expressed in probabilistic terms:
where P_{ ϖ }is the joint probability to have i and j aligned in a given alignment and P_{ π }the measure of probability that amino acids occur in a given sequence. From equations (4) and (6), equation (7) becomes:
s(i, j) = h(i) + h(j)  h(i ∩ j) (8)
that is
s(i, j) = I(i; j) (9)
As a consequence, the similarity function (or score) is the mutual information between two amino acids. Additionally, the score between sequences (the sum of elementary scores between amino acids, [32, 33, 41, 42]) is, according to the hypothesis of independence of amino acid positions, the estimated mutual information between the two given biological sequences.
Once two sequences are aligned, we pose the question whether the alignment score is sufficient to assess that the proteins are conceivably alike and thus evolutionarily related? The theorem of the upper limit of a sequence alignment score probability (TULIP theorem, [39]), sets the upper bound of an alignment score probability, under a hypothesis less restrictive than the KarlinAltschul model [43]. Given two real sequences a and b (a = a_{1}a_{2}...a_{ m }and b = b_{1}b_{2}...b_{ n }), where s = s(a,b) the maximal score of a pairwise alignment obtained with any alignment method, b* the variable corresponding to the shuffled sequences from b, and given P{S(a,b*)≥s} the probability that an alignment by chance between a and b* has a higher score than s, then whatever the distribution of the random variable S(a,b*) the TULIP theorem states:
with k > 1, μ the mean of and σ its standard deviation. The unique restriction on S(a,b*) is that it has a finite mean and a finite variance. A first corollary of the TULIP theorem is that the Zscore is a statistical test for the probability of a sequence alignment score. We additionally state the following new corollary.
TULIP corollary 2
The best upper bound value for P{S(a,b*)≥s} is termed . From the TULIP theorem and corollaries, the comparison of a protein to a given reference a, weighed by an alignment score, is characterized by a bounded probability that the alignment is fortuitous.
Question of the proximity between protein sequences in the light of information theory
Since the optimized alignment score of two protein sequences allows an access to both the mutual information between proteins and an upper bound that the alignment is not fortuitous, one would expect that it is an accurate way to spatially organize proteins sets. A simple relation would be "the higher the mutual information, the nearest". There are three ways to assess the proximity between two objects a and b in a given space E [41]. The first is dissimilarity, a function f(a,b): E × E → ^{+} such that f(a,b) = 0 ⇔ a = b and f(a,b) = f(b,a); the second is the distance per se, that is a dissimilarity such that the triangle inequality is respected: ∀ a,b,c ∈ E, f(a,c) ≤ f(a,b) + f(b,c); and the third is the similarity defined as a function f(a,b): E × E → such that and f(a,b) = f(b,a). Representing objects in a space is convenient using the notion of distance. When the optimal alignment is global, i.e. requiring that it extends from the beginning to the end of each sequence [32], it is theoretically possible to define a distance per se, that is to spatially organize the compared sequences [41]. However, from a biological point of view, global alignment algorithms are not reliable to assess homology of protein domains. Local alignments are better suited, using scoring matrices to find the optimum local alignment and maximizing the sum of the scores of aligned residues [28, 31]. In contrast with global alignments, local alignments do not allow any trivial definition of distances [41].
Although amino acid similarity is a function f(i, j): E × E → , owing to the local alignment optimization algorithms, the computed score is a function f(a,b): E × E → ^{+}, requiring the existence of at least one positive score in the similarity matrices. Thus, when constructing an alignment with the Smith and Waterman [33] method, the constraint that s(a,b)>0 (i.e. I(a;b) > 0) is imposed. This condition is consistent with proposition 1: if two sequences are homologous, knowledge about the first has to bring information about the second, that is to say, the mutual information between the two sequences cannot decrease below zero: I(a;b) > 0 (i.e. s(a,b) > 0). As a consequence, in the following geometric construction we sought a refined expression for the proximity of proteins.
Geometric construction of a configuration space of homologous proteins (CSHP) conserving mutual information
In a set of homologous proteins, any sequence a can be selected as a reference, noted a_{ ref }, in respect to which the others are compared. A geometric representation of objects relatively to a fixed frame is known as a configuration space (CS). In physics, a CS is a convenient way to represent systems of particles, defined by their positional vectors in some reference frame. Here, given n similar sequences, it is therefore possible to consider n references of the CSHP. In a given (CSHP, a_{ ref }), each amino acid position aligned with a position in the a_{ ref }sequence, corresponds to a comparison dimension (CS dimension). Proteins are simply positioned by a vector, the coordinates of which are given by the scores of aligned amino acids. Gaps are additional dimensions of the CS. When considering that local algorithms identify the space of biological interest, i.e. a CSHP, the gap penalty is a parameter that maximizes the shared informative dimensions. Thus, given the amino acids mutual information, alignment optimization methods define the relative positions of proteins.
At this point in our construction, a first important property of the CSHP can be deduced. Since mutual information with a_{ ref }is sufficient for the full positioning, then positioning of proteins in a given (CSHP, a_{ ref }) is unambiguous, unique, and is not altered when proteins are added or removed. In other words, a (CSHP, a_{ ref }) is a univocal space.
Given two sequences a and b, if b occurs in (CSHP, a_{ ref }), then a also occurs in (CSHP, b_{ ref }). The pairwise alignment of a and b having no order (symmetry of the mutual information), the positions of b in (CSHP, a_{ ref }) is dependent of the position of a in (CSHP, b_{ ref }). Thus, once a (CSHP, a_{ ref }) has been built, ∀b ∈ (CSHP, a_{ ref }), part of the geometry of (CSHP, b_{ ref }) is learnt. Thus, in a CSHP, information needed for the position of n sequences is totally contained in the geometry of the n (CSHP, a_{ ref }). This geometric stability is not observed with multiple alignments, which can be deeply modified by addition or removal of sequences. In the CSHP, protein position is unaltered by additions or removals of other proteins. In practice, the construction of CSHP is therefore completely deduced from any allbyall protein sequence comparison [45, 46] and can be easily updated.
The qdissimilarity, a proximity notion for a geometric representation of the CSHP
In the CSHP, the definition of a distance per se based on mutual information is reduced ad absurdum (For demonstration, see methods). To define a proximity function i) sharing properties of distance, i.e. increasing when objects are further apart, ii) deriving from similarity and iii) relying on mutual information, particularly the property "f(a,a) ≠ f(b,b) is possible", we introduce a fourth notion of proximity. Such proximity was called qdissimilarity (for quasidissimilarity), a function f(a,b): E × E → ^{+} is defined such that
∀ a ∈ E, ∀ b ∈ E, f(a,b) = f(b,a) (13)
Let s be a similarity, then q = e^{s}is a qdissimilarity, named the 'canonical qdissimilarity' associated to s. Accordingly, the TULIP theorem allows a statistical characterization of q(a,b) the canonical qdissimilarity between two sequences a and b.
TULIP corollary 3
From the TULIP corollary 2, relation (14) is simply deduced:
with Q(a,b*) being the random qdissimilarity variable associated with S(a,b*). Given a (CSHP, a_{ ref }), each sequence b aligned with a is characterized by a qdissimilarity q(a,b). In geometric terms, b can be represented as a point contained in a hypersphere B of radius q(a,b).
Unification of pairwise alignments theory, information theory, pdistance and qdissimilarity in the CSHP model
A geometric space is a topological space when endowed with characterized paths that link its elements. Here, paths can be defined as the underlying evolutionary history separating sequences [11]. Given u the common unknown ancestor, then the divergence time t(a,b) is theoretically the summed elapsed times separating u to a and to b. Without any empirical knowledge of u, the simplest approximation for t(a,b) was sought as a function of the fraction of identical residues f_{ id }, thus of the pdistance. With the hypothesis of the molecular clock, this function can be given as equation (2), where the transmutation of a and b is a consequence of a Poisson process. By using relation (9) on the equivalence between score similarity and mutual information, then the fundamental postulate "the closer in the evolution, the more alike and conversely, the more alike, probably the closer in the evolution" can be reformulated:
Fundamental postulate
Given two homologous proteins a and b, the closer in the evolution, the greater the mutual information between a and b (i.e. the optimal computed score s(a,b)) and conversely, the greater the mutual information between a and b, probably the closer in the evolution.
Whereas the first part of the postulate is a consequence of the conservational pressure on mutual information, the second assertion founds the historical reconstruction underlying a set of biological sequences on statistical concepts. A corollary is that evolution of two homologous proteins is characterized by a loss of mutual information.
In the CSHP, this formulation of the fundamental postulate allows a novel mathematical formalization of the pdistance in probabilistic terms. Basically, the pdistance is the divergence observed between two sequences knowing that they share some features (the observed sequences a and b) and that they were identical before the speciation event (sequence u).
Looking back to equation (1), we can reformulate f_{ id }in probabilistic terms, considering the fraction of shared features (identical sites) knowing the observed data and the existence of a common ancestor. Given two proteins a and b, let us consider the random variable Q(a,b*), defined in TULIP corollary 3. In (CSHP, a_{ ref }), shown in Figure 1, one can define the probability law P{Q(a,b*)≤ρ} as the probability that the qdissimilarity between b* and a_{ ref }is lower than ρ. The hypersphere of radius ρ contains therefore the b* random sequences sharing informative features with a accordingly. The probability p_{id/a}that b* shares identity with a, knowing that the qdissimilarity between b* and a is lower than that between the real sequences b and a, is:
p_{id/a}(b*) = P{Q(a,b*) ≤ q(a,a) / Q(a,b*) ≤ q(a,b)} (15)
which is a probabilistic expression of f_{ id }in respect to the reference a_{ ref }. According to the Venn diagram in Figure 1: p_{id/a}(b*) = P(A/B)
Using the Bayes theorem, equation (15) can be expressed as:
In consequence:
which can be expressed as
Assuming that substitution rates are independent of lineages [35], then random sequence models a* and b* are equivalent, that is to say Q(a,a*) ≈ Q(a,b*) and
Thus p_{id/a}, and symmetrically p_{id/b}, provide a probabilistic expression of f_{ id }knowing the data, i.e. the observed mutual information between a and b expressed as Q(a,b).
Given two homologous sequences a and b, when their optimal score is s(a,b) ≥ μ + ψ with ψ being a critical threshold value depending on the score distribution law (See Methods for the demonstration for the critical threshold), owing to the TULIP corollary 2, we can state that p_{id/a}is bounded above:
This expression can also be developed as:
where μ_{1}, σ_{1}, μ_{2} and σ_{2} are the mean and the standard deviation of S(a,b*) and S(a,a*) respectively. The right term in relation (21) exhibits analogies with f_{ id }given by equation (1), showing that the pragmatic approach by Feng and Doolittle [19] could be supported and generalized in a theoretical elaboration.
Using the Poisson correction, an expression of t(a,b) is given as the linear combination of the two corrections of the pdistance deduced from p_{id/a}and p_{id/b}:
t(a,b) = [log(p_{id/a}(b*)) +log(p_{id/b}(a*))] (22)
with a* and b* the random variables corresponding to the shuffled sequences of a and b respectively. The sum of the logarithms corresponds to the product of the two probabilities, an expression of the hypothesis of independence of lineage. Interestingly, equation (22) provides an expression of the symmetric effect of time on the variations that independently affected a and b.
From relation (20), t(a,b) appears as a function of Zscore ratios. For any set of homologous proteins, it is therefore possible to measure a table of pairwise divergence times and build phylogenetic trees using distance methods.
Reconstruction of protein phylogeny: first example, case study of the glucose6phosphate isomerase phylogeny
We compared the trees we obtained, called TULIP trees, to phylogenetic trees built using classical methods, for instance the popular PHYLIP [47] or PUZZLEbased [48] methods, termed here MAB trees (for multiple alignmentbased trees). Firstly, because MAB trees are constructed from multiple alignments, removals or additions of proteins modify the multiple alignments. Inclusion of sequences is considered as a way to improve the quality of multiple alignments and to increase the sensitivity of the comparison of distant sequences [49, 50]. By contrast, the protein space used to build TULIP trees is not reordered when data sets are incremented or decremented (drawing of the TULIP tree may apparently change due to the tree graphic representation methods; nevertheless the absolute tree topology is not reordered). This remarkable property is due to both the geometrical construction by pairwise comparison and the convergence of the distance matrix elements estimated by equation (21). Indeed, the estimate of the righthand term of equation (21) relies on a Monte Carlo method, after randomization of the biological sequences [39, 44, 51] and is therefore dependent on the sequence randomization model [52] and convergent in respect to the weak law of large numbers [53]. Convergence is proportional to , where numb_{ rand }is the number of randomizations. In the case studies presented here, we set num_{ rand }= 2000 (see Methods). By contrast, stability of MAB trees is sought by bootstrapping approaches and consensus tree reconstruction. MAB trees appear as the result of a complex learning process including possible readjustment of the multiple alignments after eye inspection pragmatically applied to assist the reconstruction. Alternatively, Bayesian analyses have been recently proposed for phylogenetic inference [54], estimating posterior probability of each clades to assess most likely trees. Still, in a recent comparative study, Suzuki et al. [55] and Simmons et al. [56] provided evidence supporting the use of relatively conservative bootstrap and jacknife approaches rather than the more extreme overestimates provided by the Markov Chain Monte Carlobased Bayesian methods. In the absence of any decisive methods to assess the validity of the trees obtained after such different approaches, no absolute comparison with the TULIP classification trees can be rigorously provided.
Reconstruction of protein phylogeny: second example, case study of the enolase phylogenic incongruence
Based on our theoretical model, we constructed the corresponding TULIP tree. TULIP trees given with BLOSUM 62 or PAM 250 matrices, FitchMargoliash or neighborjoining methods led indistinctly to a unique tree topology (Figure 4B). Separation of great phyla (Archaebacteria, Eubacteria, Diplomonads, Trypanasomes, Animals, Fungi and Amoeba) is recovered. A plantlike cluster is additionally reconstructed, in which a distinct separation occurred between {Rhodophytes ; Cryptomonads} and {Land Plants ; Charophytes ; Chlorarachnion ; Alveolates} main clusters. It is remarkable that this latter cluster is that characterized by the enolase insertion.
This topology corresponds to the observed distribution of the enolase short insertions and provides therefore a solution to the apparent enolase phylogeny incongruence: the phylogenetic position of alveolates is not in conflict with the distribution of enolase insertion and the apicomplexa enolase is possibly a consequence of a lateral transfer, like in cryptomonads.
Large scale phylogeny based on a CSHP built from massive genomic pairwise comparisons
A CSHP containing large sets of protein sequences can be built after any allbyall massive comparison providing Zscore statistics. Because the space elaboration is explicit, then quality of the mutual information conservation depends on the choice of the scoring matrix, the geometric positioning depends on the local alignment method, the homology assessment depends on the alignment score and probabilistic cutoffs and the phylogenetic topology on the choice of the stochastic law correction. Eventually, genomescale pairwise comparisons [39, 36] find in the present CSHP a robust, evolutionary consistent and easily updatable representation.
Methods
Glucose6Phosphate Isomerase sequences
The 41 Glucose6phosphate isomerase (EC 5.3.1.9) sequences studied in the paper are taken from several representative groups, as provided from the Swissprot database. Group I: Archae ([Swissprot:G6PI_HALN1], Halobacterium sp.; [Swissprot:G6PI_METJA], Methanococcus jannaschii). Group II: Bacteria Actinobacteria ([Swissprot:G6P1_STRCO], Streptomyces coelicolor; [Swissprot:G6PI_COREF, Corynebacterium efficiens; [Swissprot:G6PI_MYCTU], Mycobacterium tuberculosis). Group III: Bacteria Cyanobacteria ([Swissprot:G6PI_ANASP], Anabaena sp.; [Swissprot:G6PI_SYNEL], Synechococcus elongates). Group III: Bacteria Bacillus ([Swissprot:G6PI_LACFE], Lactobacillus fermentum; [Swissprot:G6PI_BACHD], Bacillus halodurans; [Swissprot:G6PI_BACSU], Bacillus subtilis; [Swissprot:G6PI_CLOPE], Clostridium perfringens). Group IV: Bacteria Proteobacteria ([Swissprot:G6PI_BIFLO], Bifidobacterium longum; [Swissprot:G6PI_ECOLI], Escherichia coli). Group V: Bacteria Chlamydiae ([Swissprot:G6PI_CHLTR], Chlamydia trachomatis; [Swissprot:G6PI_CHLCV], Chlamydophila caviae; [Swissprot:G6PI_CHLMU], Chlamydia muridarum). Group VI: Others Bacteria ([Swissprot:G6PI_CHLTE], Chlorobium tepidum; [Swissprot:G6PI_DEIRA], Deinococcus radiodurans; [Swissprot:G6PI_BORBU], Borrelia burgdorferi; [Swissprot:G6PI_THEMA], Thermotoga maritime). Group VII: Fungi ([Swissprot:G6PI_SCHPO], Schizosaccharomyces pombe; [Swissprot:G6PI_YEAST], Saccharomyces cerevisiae; [Swissprot:G6PI_NEUCR], Neurospora crassa; [Swissprot:G6PI_ASPOR], Aspergillus oryzae). Group VII: Eukaryota Viridiplantae ([Swissprot:G6PI_ARATH], Arabidopsis thaliana; [Swissprot:G6PI_MAIZE], Zea mays; [Swissprot:G6PI_SPIOL, Spinacia oleracea; [Swissprot:G6PA_ORYSA], Oryza sativa). Group VIII: Eukaryota Alveolata Apicomplexa ([Swissprot:G6PI_PLAFA], Plasmodium falciparum; [Swissprot:Q9XY88], Toxoplasma Gondii; [Swissprot:269_185], Cryptosporidium parvum). Group IX: Animals ([Swissprot:G6PI_DROME, Drosophila melanogaster; [Swissprot:G6PI_MOUSE], Mus musculus; [Swissprot:G6PI_HUMAN], Homo sapiens; [Swissprot:G6PI_PIG], Sus scrofa; [Swissprot:G6PI_RABIT], Oryctolagus cuniculus; [Swissprot:G6PI_TRYBB], Trypanosoma brucei brucei). Group X: Other Eukaryota ([Swissprot:AY581147], Entamoeba histolytica; [Swissprot:G6PI_LEIME], Leishmania mexicana; [Swissprot:AY581146], Dictyostelium discoideum; [Swissprot:Q968V7], Giardia intestinalis).
Enolase sequences
Enolase sequences used for the casestudy presented in this paper were taken from eight major groups previously studied by [36]. Group I: Land Plant, Charophytes, Chlorophytes, Rhodophytes and Cryptomonads ([Swissprot:CAA39454], Zea mays; [Swissprot:Q42971], Oryza sativa; [Swissprot:Q43130], Mesembryanthemum crystallinum; [Swissprot:P42896], Ricinus communis; [Swissprot:Q43321], Alnus glutinosa; [Swissprot:Q9LEJ0], Hevea brasiliensis 1; [Swissprot:P25696], Arabidopsis thaliana; [Swissprot:P26300], Lycopersicon esculentum; [Swissprot:AF348914], Chara corallina; [Swissprot:AF348915], Nitella opaca; [Swissprot:AF348916], Nitellopsis obtusa; [Swissprot:AF348918], Pycnococcus provasolii 2; [Swissprot:AF348919], Bigelowiella natans – Chlorarachnion ; [Swissprot:AF348920], Mastocarpus papillatus 1; [Swissprot:AF348923], Prionitis lanceolata 1; [Swissprot:AF348931], Rhodomonas salina 1; [Swissprot:AF348933], Guillardia theta; [Swissprot:AF348935], Pedinomonas minor). Group II : Animals and Fungi ([Swissprot:P04764], Rattus norvegicus; [Swissprot:P51913], Gallus gallus A; [Swissprot:P07322], Gallus gallus B; [Swissprot:Q9PVK2], Alligator mississippiensis; [Swissprot:P06733], Homo sapiens A; [Swissprot:P13929, Homo sapiens B; [Swissprot:P15007], Drosophila melanogaster; [Swissprot:AF025805], Drosophila pseudoobscura; [Swissprot:O02654], Loligo pealeii; [Swissprot:AF100985], Penaeus monodon; [Swissprot:Q27527], Caenorhabditis elegans; [Swissprot:Q27877], Schistosoma mansoni; [Swissprot:P33676], Schistosoma japonicum; [Swissprot:Q27655], Fasciola hepatica; [Swissprot:P00924], Saccharomyces cerevisiae 1; [Swissprot:Q12560], Aspergillus oryzae; [Swissprot:P42040], Cladosporium herbarum; [Swissprot:P40370], Schizosaccharomyces pombe 1; [Swissprot:AF063247], Pneumocystis carinii f.). Group III: Amoebae ([Swissprot:P51555], Entamoeba histolytica; [Swissprot:Q9U615], Mastigamoeba balamuthi). Group IV: Alveolates ([Swissprot:AF348926], Paramecium multimicronucleatum; [Swissprot:AF348927], Paramecium tetraurelia; [Swissprot:AF348928], Colpidium aqueous; [Swissprot:AF348929], Tetrahymena thermophila I; [Swissprot:AF348930], Tetrahymena bergeri; [Swissprot:Q27727], Plasmodium falciparum; [Swissprot:AF051910], Toxoplasma gondii). Group V: Trypanosomatidae ([Swissprot:AF159530], Trypanosoma cruzi eno1 partial; [Swissprot:AF152348], Trypanosoma brucei complete). Group VI: Hexamitidae ([Swissprot:AF159519], Hexamita inflata eno1 partial; [Swissprot:AF159517], Spironucleus vortens partial). Group VII: Archaebacteria ([Swissprot:Q9UXZ0], Pyrococcus abyssi; [Swissprot:O59605], Pyrococcus horikoshi i; [Swissprot:Q60173], Methanococcus jannaschii; [Swissprot:Q9Y927], Aeropyrum pernix). Group VII: Eubacteria ([Swissprot:O66778], Aquifex aeolicus; [Swissprot:P37869],Bacillus subtilis; [Swissprot:Q9K717], Bacillus halodurans; [Swissprot:P77972], Synechocystis sp.; [Swissprot:P33675], Zymomonas mobilis; [Swissprot:P08324], Escherichia coli; [Swissprot:P47647], Mycoplasma genitalium; [Swissprot:Q8EW32, Mycoplasma penetrans; [Swissprot:P74934], Treponema pallidum).
Demonstration that distance of a protein to itself cannot be defined in the CSHP
In the simplest case, building a distance between amino acids (that would lead to distance between sequences) on the basis of computed similarity values would have to respect the condition:
∀i ∈ E, ∀j ∈ E,d(i,j) = 0 ⇒ i = j (a)
for i and j, two given amino acids and d the distance function. Using this condition in the proposition, any organization of the CSHP with a geometric distance is reduced ad absurdum.
Proposition
Building a distance between amino acids derived from the composed function d(i,j) = (φ ○ s)(i,j), where s is a similarity function and φ a bijection, is impossible without a loss of mutual information. Moreover, two proteins from distinct organisms can have the same configuration, being like "twins", and d(i,j) = 0 does not imply i = j.
Proof
Condition (a) implies that φ (s(i,i)) = φ (s(j,j)) = 0. This equality imposes that s(i,i) = s (j,j) and, following equation (7) of main text, that I(i;i) = I(j;j). Considering for example tryptophan (W) and glutamic acid (E), if W occurs in a sequence, the mutual information gained about the occurrence of W at the aligned position would be the same as that gained in the case of E about the occurrence of E at the aligned position in the homologous protein. This statement is easily rejected on the basis of biochemical concerns. On one hand, aspartic acid (D) shares common biochemical properties with E, particularly a carboxylic acid, and easily substitutes in homologous sequences. By contrast W, exhibiting a unique biochemical feature, is less substitutable without altering the function. Thus the mutual information I(E;E) is necessarily lower than I(W;W). This that can be checked in scoring matrices such as BLOSUM 62 [30] where I(E;E) = 5, I(D;D) = 6 and I(W;W) = 11. Condition d(i,i) = 0 leads to an obvious loss of information. The second assertion of the proposition is obvious.
Determination of the threshold value ψ, for topological reconstructions in the CHSP based on pairwise alignment score probabilities
An important basis of the reconstruction of a probabilistic evolutionary topology in the CSHP is based on the demonstration that, given S the random variable corresponding to the alignment scores of pairs of shuffled sequences and μ the mean of S, given two homologous sequences a and b, when their optimal score is s(a,b) ≥ μ + ψ (with ψ a critical threshold value depending on the score distribution law), owing to the TULIP corollary 2, we can state that p_{id/a}is bounded above
To the purpose of this demonstration, we considered the cumulative distribution function F(s) = P(S ≤ s), its derivative f(s) known as the probability density function defined as dF(s) = f(s)ds, and the positive delta function δ (s) = (s  μ)^{2}(1  F(s)). Since δ (s) = (s  μ)^{2}(1  F(s)) is null for s = μ and , the Rolle's theorem implies that ∃s_{0} ∈]μ,+∞[ such as [60]; s_{0} corresponds to a maximum of δ (s) and is therefore the solution of the equation
2(1  F(s))  (s  μ) f(s) = 0 (b)
one can express as
In consequence, δ (s) reaches its maximum for a s_{0} (s_{0} ≤ μ + ψ) and it is strictly decreasing on ]μ + ψ ,+∞[.
The estimation of s_{0} is not trivial because it depends on the knowledge of the cumulative distribution function. Extensive studies provided experimental and theoretical supports for an extreme value distribution of alignment scores [31, 43, 44]. Using the extreme value distribution of type I, i.e. the Gumbel distribution [62], the cumulative distribution is given by
with θ and β (β > 0) the location and scale parameters. The probability density function g(s) is defined by dG(s) = g(s)ds. We observe with that . Using the Taylor's polynomial formula, i.e. :
In consequence, for a Gumbel score probability distribution:
If a pairwise alignment score of two sequences a and b is relatively high, that is s(a,b) ≥ μ + ψ, then the trivial inequality s(a,a) ≥ s(a,b) implies
(s(a,b)  μ)^{2}(1  F(s(a,b))) ≥ (s(a,a)  μ)^{2}(1  F(s(a,a))) (h)
that is to say
From inequality (i), we deduce that p_{id/a}is bounded above.
Construction of PHYLIP multiple alignment based trees and pairwise alignment based TULIP trees
To build PHYLIP trees, multiple sequence alignments were created with ClustalW [63]. PHYLIP trees where constructed using the protpars and neighbor modules from the PHYLIP package [47] and the BLOSUM 62 substitution matrix. Bootstrap support was estimated using 1000 replicates. To build TULIP trees, for each couple of sequences a and b, alignment was achieved with the SmithWaterman method and the BLOSUM 62 scoring matrices, using the BIOFACET package from GeneIT, France [64]. We computed estimated zscores z(a,b*), z(a,a*), z(a*,b), z(b*,b*), with 2000 sequence shuffling. For all computations, an estimation of the Gumbel parameters θ and β was made using the computed μ and σ of any S(a,b*) and the formula and θ = μ  β Γ'(1), where Γ'(1) ≈ 0.577216 is the Euler constant. In all computations, both Gumbel parameters were very close (in the case of enolases, mean(θ) = 35.04, SD(θ) = 0.12, mean(β) = 3.92, SD(β) = 0.08). As a consequence, the assumption Q(a,a*) ≈ Q(a,b*) was verified for any pair of sequences. We used the parameters to estimate μ = θ + β Γ '(1) (in the case of enolases, μ = 37.33), and μ + ψ ≈ μ + 10.5178 ≈ 47.85. As any pairs of computed scores are higher than this critical threshold, we used relation [20]. Estimation of evolutionary time was achieved according to equations [20] and [22]. Trees were constructed using FitchMargoliash and NeighborJoining methods [47].
List of abbreviations
 CSHP:

configuration space of homologous proteins, TULIP, theorem of the upper limit of a score probability
Declarations
Acknowledgements
We thank John Dunlop for copy editing and Joël Bleuse and Jacques Demongeot for critical reading of this manuscript. We are indebted to JeanPaul Eynard, JeanMichel Pabiot and JeanJacques Codani for supportive help. Part of this work was financed by Ministère de la Recherche et de la Technologie and by Agence Nationale de la Valorisation de la Recherche RhôneAlpes.
Authors’ Affiliations
References
 Zuckerkandl E, Pauling L: Molecules as documents of evolutionary history. J Theor Biol 1965, 8: 357–366.View ArticlePubMedGoogle Scholar
 Zukerkandl E: The evolution of hemoglobin. Sci Am 1965, 212: 110–118.View ArticleGoogle Scholar
 Fitch WM, Margoliash E: Construction of phylogenetic trees. Science 1967, 155: 279–284.View ArticlePubMedGoogle Scholar
 Arnheim N, Taylor CE: NonDarwinian evolution: consequences for neutral allelic variation. Nature 1969, 223: 900–903.View ArticlePubMedGoogle Scholar
 Dayhoff MO: Computer analysis of protein evolution. Sci Am 1969, 221: 86–95.View ArticlePubMedGoogle Scholar
 Arnheim N, Steller R: Multiple genes for lysozyme in birds. Arch Biochem Biophys 1970, 141: 656–661. 10.1016/00039861(70)901852View ArticlePubMedGoogle Scholar
 DeLange RJ, Smith EL: Histones: structure and function. Annu Rev Biochem 1971, 40: 279–314. 10.1146/annurev.bi.40.070171.001431View ArticlePubMedGoogle Scholar
 Zuckerkandl E: Some aspects of protein evolution. Biochimie 1972, 54: 1095–102.View ArticlePubMedGoogle Scholar
 Dayhoff MO, Barker WC, McLaughlin PJ: Inferences from protein and nucleic acid sequences: early molecular evolution, divergence of kingdoms and rates of change. Orig Life 1974, 5: 311–330. 10.1007/BF01207633View ArticlePubMedGoogle Scholar
 Wu TT, Fitch WM, Margoliash E: The information content of protein amino acid sequences. Annu Rev Biochem 1974, 43: 539–566. 10.1146/annurev.bi.43.070174.002543View ArticlePubMedGoogle Scholar
 Brocchieri L: Phylogenetic inferences from molecular sequences: review and critique. Theor Popul Biol 2001, 59: 27–40. 10.1006/tpbi.2000.1485View ArticlePubMedGoogle Scholar
 Singer GA, Hickey DA: Nucleotide bias causes a genomewide bias in the amino acid composition of proteins. Mol Biol Evol 2000, 17: 1581–1588.View ArticlePubMedGoogle Scholar
 Bastien O, Lespinats S, Roy S, Metayer K, Fertil B, Codani JJ, Maréchal E: Analysis of the compositional biases in Plasmodium falciparum genome and proteome using Arabidopsis thaliana as a reference. Gene 2004, 336: 163–173. 10.1016/j.gene.2004.04.029View ArticlePubMedGoogle Scholar
 Doolittle RF: Similar amino acid sequences: chance or common ancestry? Science 1981, 214: 149–159.View ArticlePubMedGoogle Scholar
 Otu HH, Sayood K: A new sequence distance measure for phylogenetic tree construction. Bioinformatics 2003, 19: 2122–2130. 10.1093/bioinformatics/btg295View ArticlePubMedGoogle Scholar
 Jukes TH, Cantor CR: Mammalian Protein Metabolism. New York: Academic Press; 1969.Google Scholar
 Kimura M: A simple model for estimating evolutionary rates of base substitiutions through comparative studies of nucleotide sequences. J Mol Evol 1980, 16: 111–120.View ArticlePubMedGoogle Scholar
 Lake JA: Reconstructing evolutionary trees from DNA and protein sequences: paralinear distances. Proc Natl Acad Sci USA 1994, 91: 1455–1459.PubMed CentralView ArticlePubMedGoogle Scholar
 Feng DF, Doolittle RF: Converting amino acid alignment scores into measures of evolutionary time: a simulation study of various relationships. J Mol Evol 1997, 44: 361–370.View ArticlePubMedGoogle Scholar
 Camin J, Sokal R: A method for deducing branching sequences in phylogeny. Evolution 1965, 19: 311–326.View ArticleGoogle Scholar
 Fitch WM: Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 1971, 35: 406–416.View ArticleGoogle Scholar
 Felsenstein J: Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 1981, 17: 368–376.View ArticlePubMedGoogle Scholar
 Felsenstein J, Churchill GA: A hidden Markov model approach to variation among sites in rate of evolution. Mol Biol Evol 1996, 13: 93–104.View ArticlePubMedGoogle Scholar
 Salemi M, Vandamme AM: The Phylogenetic Handbook. Cambridge University Press; 2003.Google Scholar
 Feng DF, Cho G, Doolittle RF: Determining divergence times with a protein clock: update and reevaluation. Proc Natl Acad Sci USA 1997, 94: 13028–13033. 10.1073/pnas.94.24.13028PubMed CentralView ArticlePubMedGoogle Scholar
 Nei M, Xu P, Glazko G: Estimation of divergence times from multiprotein sequences for a few mammalian species and several distantly related organisms. Proc Natl Acad Sci USA 2001, 98: 2497–2502. 10.1073/pnas.051611498PubMed CentralView ArticlePubMedGoogle Scholar
 Doolittle RF, Feng DF, Tsang S, Cho G, Little E: Determining divergence times of the major kingdoms of living organisms with a protein clock. Science 1996, 271: 470–477.View ArticlePubMedGoogle Scholar
 Dayhoff MO, Barker WC, Hunt LT: Establishing homologies in protein sequences. Methods Enzymol 1983, 91: 524–545.View ArticlePubMedGoogle Scholar
 Risler JL, Delorme MO, Delacroix H, Henaut A: Amino acid substitutions in structurally related proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix. J Mol Biol 1988, 204: 1019–1029. 10.1016/00222836(88)900587View ArticlePubMedGoogle Scholar
 Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 1992, 89: 10915–10919.PubMed CentralView ArticlePubMedGoogle Scholar
 Waterman MS: Introduction to computational biology. CRC Press; 1995.View ArticleGoogle Scholar
 Needleman SB, Wunsch CD: A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 1970, 48: 443–453. 10.1016/00222836(70)900574View ArticlePubMedGoogle Scholar
 Smith TF, Waterman MS: Identification of common molecular subsequences. J Mol Biol 1981, 147: 195–197. 10.1016/00222836(81)900875View ArticlePubMedGoogle Scholar
 Fitch WM: Random sequences. J Mol Biol 1983, 163: 171–176. 10.1016/00222836(83)900025View ArticlePubMedGoogle Scholar
 Grishin NV: Estimation of the number of amino acid substitutions per site when the substitution rate varies among sites. J Mol Evol 1995, 41: 675–679. 10.1007/BF00175826View ArticlePubMedGoogle Scholar
 Keeling PJ, Palmer JD: Lateral transfer at the gene and subgenic levels in the evolution of eukaryotic enolase. Proc Natl Acad Sci USA 2001, 98: 10745–10750. 10.1073/pnas.191337098PubMed CentralView ArticlePubMedGoogle Scholar
 Hartley RVL: Transmission of Information. The Bell System Technical Journal 1928, 3: 535–564.View ArticleGoogle Scholar
 Shannon CE: A Mathematical Theory of Communication. The Bell System Technical Journal 1948, 27: 379–423.View ArticleGoogle Scholar
 Bastien O, Aude JC, Roy S, Maréchal E: Fundamentals of massive automatic pairwise alignments of protein sequences: theoretical significance of Zvalue statistics. Bioinformatics 2004, 20: 534–537. 10.1093/bioinformatics/btg440View ArticlePubMedGoogle Scholar
 Dayhoff MO, Schwartz RM, Orcutt BC: A Model of Evolutionary Change in Proteins. Atlas of Protein Sequence and Structure 1978, 5: 345–352.Google Scholar
 Setubal J, Meidanis J: Introduction to Computational Molecular Biology. PWS Publishing Compagny; 1997.Google Scholar
 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215: 403–410. 10.1006/jmbi.1990.9999View ArticlePubMedGoogle Scholar
 Karlin S, Altschul SF: Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA 1990, 87: 2264–2268.PubMed CentralView ArticlePubMedGoogle Scholar
 Comet JP, Aude JC, Glemet E, Risler JL, Henaut A, Slonimski PP, Codani JJ: Significance of Zvalue statistics of SmithWaterman scores for protein alignments. Comput Chem 1999, 23: 317–331. 10.1016/S00978485(99)00008XView ArticlePubMedGoogle Scholar
 Bacro JN, Comet JP: Sequence alignment: an approximation law for the Zvalue with applications to databank scanning. Comput Chem 2001, 25: 401–410. 10.1016/S00978485(01)000742View ArticlePubMedGoogle Scholar
 Louis A, Ollivier E, Aude JC, Risler JL: Massive sequence comparisons as a help in annotating genomic sequences. Genome Res 2001, 11: 1296–1303. 10.1101/gr.GR1776RPubMed CentralView ArticlePubMedGoogle Scholar
 Felsenstein J: PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 1989, 5: 164–166.Google Scholar
 Schmidt HA, Strimmer K, Vingron M, von Haeseler A: TREEPUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 2002, 18: 502–504. 10.1093/bioinformatics/18.3.502View ArticlePubMedGoogle Scholar
 Thompson JD, Plewniak F, Poch O: A comprehensive comparison of multiple sequence alignment programs. Nucleic Acids Res 1999, 27: 2682–2690. 10.1093/nar/27.13.2682PubMed CentralView ArticlePubMedGoogle Scholar
 Simmons MP, Freudenstein JV: The effects of increasing genetic distance on alignment of, and tree construction from, rDNA internal transcribed spacer sequences. Mol Phylogenet Evol 2003, 26: 444–451. 10.1016/S10557903(02)003664View ArticlePubMedGoogle Scholar
 Manly BFJ: Randomization, Bootstrap and Monte Carlo Methods in Biology. CRC Press; 1997.Google Scholar
 White S: Global statistics of protein sequences: implications for the origin, evolution, and prediction of structure. Annu Rev Biophys Biomol Struct 1994, 23: 407–439. 10.1146/annurev.bb.23.060194.002203View ArticlePubMedGoogle Scholar
 Capinski M, Kopp E: Measure, Integral and Probability. NewYork: SpringerVerlag; 1999.View ArticleGoogle Scholar
 Rannala B, Yang Z: Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. J Mol Evol 1996, 43: 304–311.View ArticlePubMedGoogle Scholar
 Suzuki Y, Glazko GV, Nei M: Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proc Natl Acad Sci U S A 2002, 99: 16138–16143. 10.1073/pnas.212646199PubMed CentralView ArticlePubMedGoogle Scholar
 Simmons MP, Pickett KM, Miya M: How meaningful are Bayesian support values? Mol Biol Evol 2004, 21: 188–199. 10.1093/molbev/msh014View ArticlePubMedGoogle Scholar
 Huang J, Mullapudi N, Lancto CA, Scott M, Abrahamsen MS, Kissinger JC: Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome Biol 2004, 5: R88. 10.1186/gb2004511r88PubMed CentralView ArticlePubMedGoogle Scholar
 Read M, Hicks KE, Sims PF, Hyde JE: Molecular characterisation of the enolase gene from the human malaria parasite Plasmodium falciparum. Evidence for ancestry within a photosynthetic lineage. Eur J Biochem 1994, 220: 513–520. 10.1111/j.14321033.1994.tb18650.xView ArticlePubMedGoogle Scholar
 Dzierszinski F, Popescu O, Toursel C, Slomianny C, Yahiaoui B, Tomavo S: The protozoan parasite Toxoplasma gondii expresses two functional plantlike glycolytic enzymes. Implications for evolutionary origin of apicomplexans. J Biol Chem 1999, 274: 24888–24895. 10.1074/jbc.274.35.24888View ArticlePubMedGoogle Scholar
 Lang S: Undergraduate analysis. NewYork: SpringerVerlag; 1997.View ArticleGoogle Scholar
 Valleron AJ: Introduction à la Biostatistique. Paris: Masson; 1998.Google Scholar
 Coles S: An introduction to Statistical Modeling of Extreme Values. NewYork: SpringerVerlag; 2001.View ArticleGoogle Scholar
 Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22: 4673–4680.PubMed CentralView ArticlePubMedGoogle Scholar
 Codani JJ, Comet JP, Aude JC, Glémet E, Wozniak A, Risler JL, Hénaut A, Slonimski PP: Automatic analysis of largescale pairwize alignments of protein sequences. Methods in Microbiology 1999, 28: 229–244.View ArticleGoogle Scholar
Copyright
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.