the ever - increasing size of homologous sequence datasets and complexity of substitution models stimulate the development of better methods for building phylogenetic trees . likelihood - based approaches ( including bayesian ) provided arguably the most successful advances in this area in the last decade .
different strategies have then been used to tackle this problem , mostly based on stochastic approaches .
markov chain monte carlo methods are probably the most valuable tools in this context as they provide computationally tractable solutions to bayesian estimation of phylogenies ( 1,2 ) .
stochastic approaches have also been used to address optimization issues in the maximum likelihood framework .
hence , simulated annealing ( 3 ) and genetic algorithms ( 4,5 ) were proposed to estimate maximum likelihood phylogenies from large datasets .
however , the hill climbing principle is usually considered faster than stochastic optimization and sufficient for numerous combinatorial optimization problems ( 6 ) .
recently , guindon and gascuel ( 7 ) described a fast and simple heuristic based on this principle , for building maximum likelihood phylogenies .
several simulation studies ( 7,8 ) demonstrated that the tree topologies estimated with this approach are as accurate as those inferred using the best tree building methods currently available .
these studies also showed that this new method is considerably faster than the other likelihood - based approaches . using this heuristic ,
the analysis of large datasets is now achieved in reasonable computing time on any standard personal computer ; e.g. only 12 min were required to analyse a dataset consisting of 500 rbcl sequences with 1428 bp from plant plastids .
this paper introduces phyml online , a web interface to the phyml ( phylogenetic inferences using maximum likelihood ) software that implements the heuristic described by guindon and gascuel ( 7 ) .
phyml online provides a number of useful options ( e.g. nonparametric bootstrap ) , and proposes quite recent models of sequence evolution [ e.g. wag ( 9 ) and dcmut ( 10 ) ] .
the core of the heuristic is based on a well - known tree - swapping operation , namely 
nearest neighbour interchange , which defines three possible topological configurations around each internal branch ( 11 ) . for each of these configurations ,
the length of the internal branch that maximizes the likelihood is estimated using numerical optimization .
the difference of likelihood obtained under the best alternative topological configuration and the current one defines a score .
a score with positive value indicates that the best alternative topological configuration yields an improvement of likelihood .
a score with negative value indicates that the current topological configuration can not be improved at this stage and only the length of the internal branch is adjusted .
these calculations are performed independently for every branch and they define a set of ( topological or numerical ) modifications , each of which corresponds to an improvement of the current tree regarding the likelihood function .
the standard approach would only apply one of these modifications , typically that corresponding to the internal branch with best score . here
this proportion is adjusted so as to increase the likelihood at each step , ensuring convergence of the algorithm .
this way , the current tree is improved at each step , both in terms of topology and branch length , and only a few steps ( usually a few dozen or less ) are necessary to reach an optimum of the likelihood function .
this explains the speed of this algorithm whose time complexity is o(pns ) , where p represents the number of refinement steps that have been performed and n is the number of sequences of length s.
phyml online is a web interface to the phyml algorithm ( figure 1 ) . by default ,
the input data consists of a single text file containing one or more alignments of dna or protein sequences in phylip ( 12 ) interleaved or sequential format .
examples of sequence datasets in phylip format are given in the  user 's guide section of the web site .
setting the parameters of a phylogenetic analysis through the interface is straightforward . the first step is the selection of the substitution model of interest .
alignments of homologous dna and amino acid sequences can be examined under a wide range of models ( jc69 , k80 , f81 , f84 , hky85 , tn93 and gtr for nucleotides , and dayhoff , jtt , mtrev , wag and dcmut for amino acids ) .
variability of substitution rates across sites and invariable sites can also be taken into account .
the parameters that model the intensity of the variation of rates across sites and the proportion of invariables sites can be fixed by the user or estimated by maximum likelihood .
note that the parameters of the substitution model can be estimated under a fixed tree topology or not .
the fixed topology option is useful when describing the evolutionary process is more important than estimating the history of sequences .
an option is available to assess the reliability of internal branches using nonparametric bootstrap ( 13 ) which is possible to achieve for even large datasets , thanks to the speed of phyml optimization algorithm .
the bootstrap values are displayed on the maximum likelihood phylogeny estimated from the original dataset .
trees estimated from each bootstrap replicate , as well as the corresponding substitution parameters , can also be saved in separate files for further analysis ( e.g. computation of confidence intervals for the substitution parameters or estimation of a consensus bootstrap tree , as performed by phylip 's consense ) .
multiple trees can also be used as input and further optimized by the algorithm described above .
this might prevent the tree searching heuristic to be trapped in local maxima . when combined with the fixed tree option
, the multiple input trees approach also facilitates the comparison of the fit of different phylogenies estimated from a single dataset .
user 's guide section gives details on the format of multiple sequence and tree files .
sequences [ and starting tree(s ) if provided ] are uploaded on our server , a 16-processor ibm computer running linux 2.6.8 - 1.521custom smp , and a maximum likelihood analysis is performed using the phyml algorithm .
the first file presents a summary of the options selected by the user , maximum likelihood estimates of the parameters of the substitution model that were adjusted , and the log likelihood of the model given the data .
this applet runs the program atv ( 14 ) that provides numerous options to display and manipulate large phylogenetic trees .
the phyml online server is located at  laboratoire d'informatique , de robotique et de microlectronique de montpellier : .
the phyml software has been implemented in c ansi and is available under gnu general public licence .
binaries , example datasets , sources and documentation are distributed free of charge for academic purpose only .
 phyml online is a web interface to phyml , a software that implements a fast and accurate heuristic for estimating maximum likelihood phylogenies from dna and protein sequences . 
 this tool provides the user with a number of options , e.g. nonparametric bootstrap and estimation of various evolutionary parameters , in order to perform comprehensive phylogenetic analyses on large datasets in reasonable computing time . the server and its documentation are available at . 
INTRODUCTION
ALGORITHM
PHYML ONLINE
AVAILABILITY
Figures and Tables