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User Manual for SplitsTree4 V4.6

1. Input Characters Output Network RefinedBunemanTree This method computes the Refined Buneman Tree 3 This module was implemented by Lasse Westh Nielsen and Christian N S Pedersen Usage RefinedBunemanTree Input Distances Output Splits ReticulateNetwork This method computes a reticulate network 21 Usage ReticulateNetwork Which lt int gt Method lt String gt ShowSplits lt boolean gt DutGroup lt String gt MaxAngle lt int gt MaxReticulationsPerTangle lt int gt ShowSequences lt boolean gt ShowMutations lt boolean gt Input Splits Output Network RootedEqualAngle This method computes a rooted split network using the rooted equal angle algorithm 11 Usage RootedEqualAngle OptimizeDaylight lt boolean gt UseWeights lt boolean gt RunConvexHull lt boolean gt DaylightIterations lt int gt OutGroup lt String gt MaxAngle lt int gt SpecialSwitch lt boolean gt Input Splits Output Network RYSplits This method computes all RY splits Usage RYSplits Input Characters Output Splits SpectralSplits This method computes all splits arising using spectral analysis 14 46 Usage SpectralSplits Threshold lt double gt Method lt String gt Weight_ATvsGC lt double gt Weight_AGvsCT lt double gt Weight_ACvsGT lt double gt Input Characters Output Splits SplitDecomposition This method computes the split decomposition 1 Usage SplitDecomposition Input Distances Output
2. This method calculates distances using the Hamming distance This is identical to the UncorrectedP method Usage Hamming ignoregaps lt boolean gt Input Characters Output Distances HKY85 This method calculates distances using the Hasegawa Kishino and Yano model Usage HKY85 Estimate_Base_Frequencies lt boolean gt Normalize lt boolean gt TRatio lt double gt A lt double gt C lt double gt G lt double gt T_U lt double gt P_Invar lt double gt Gamma lt double gt Input Characters Output Distances HybridizationNetwork This method computes a hybridization network 21 The present implementation operates by calling the ReticulateNetwork method Usage HybridizationNetwork Which lt int gt Method lt String gt ShowSplits lt boolean gt DutGroup lt String gt MaxAngle lt int gt MaxReticulationsPerTangle lt int gt ShowSequences lt boolean gt ShowMutations lt boolean gt Input Splits Output Network JukesCantor This method computes distances using the Jukes Cantor model 31 Usage JukesCantor Maximum_Likelihood lt boolean gt Input Characters Output Distances K2P This method calculates distances using the Kimura 2P model 31 Usage K2P Maximum_Likelihood lt boolean gt TRatio lt double gt Input Characters Output Distances K3ST This method calculates distances using the Kimura 8ST model 31 Usage K3ST Maximum_Likelihood lt boolean gt TRatio lt double gt AC_vs_ATRatio
3. 20 6 Opening Reading and Writing Files To open a file select the File Open menu item and then browse to the desired file Alternatively if the file was recently opened by the program then it may be contained in the File Open Recent submenu The native file format of SplitsTree4 is based on the Nexus format see 25 However the program can also parse a number of other formats including ClustalW format for aligned sequences Phylip format for sequences and distances FastA format for aligned distances and Newick format for trees Earlier versions had separate Open and Import menu items these have now been combined If a file is opened while the Main Network tab or Main Data tab is open then the program will attempt to parse and execute the file If the file is a complete Nexus file previously gen erated by SplitsTree4 then the network described in the file will be displayed If the file is in some other format then depending on the type of content the program will perform a chain of default calculations If a file is opened while the Main Source tab is opened then the contents of the file is read into the program and displayed as text in the Source tab but no computation is performed To save the complete data associated with a given window in SplitsTree4 s native Nexus format use the File Save or File Save As menu items To save selected blocks of data or to export data in a different file format use the File
4. Characters Output Trees Phylogram This method computes a traditional phylogenetic tree Usage Phylogram Weights lt boolean gt Cladogram lt boolean gt Outgroup lt String gt UseOutgroup lt boolean gt Input Splits Output Network 44 PhyML This method calculates maximum likelihood trees from DNA sequences using PHYML 13 Usage PhyML PHYMLPath lt String gt TreePath lt String gt Bootstrap lt boolean gt NumberOfBootstrapReplicates lt int gt PrintBootstrap lt boolean gt SubstitutionModel lt java lang String gt OptimiseEquilibriumFrequencies lt boolean gt EquilibriumFrequenciesEmpirical lt boolean gt FrequencyA lt double gt FrequencyC lt double gt FrequencyG lt double gt FrequencyT lt double gt SubstitutionParameterAC lt int gt SubstitutionParameterAG lt int gt SubstitutionParameterAT lt int gt SubstitutionParameterCG lt int gt SubstitutionParameterCT lt int gt SubstitutionParameterGT lt int gt EmpiricalBaseFrequencyEstimates lt boolean gt GammaDistributionParameter lt double gt GammaDistributionParameterFixed lt boolean gt InvariableSitesProportion lt double gt InvariableSitesProportionFixed lt boolean gt NumberOfSubstitutionCategories lt int gt OneSubstitutionCategory lt boolean gt OptimiseStart lt boolean gt OptimiseStartKeepingTopology lt boolean gt TstvRatio lt double gt TstvRatioFixed lt boolean gt UseBioNJstart lt boolean gt OptimiseRelativeRateParameters lt bo
5. Splits SuperNetwork This method computes a super network from partial trees using the Z closure algorithm 18 Usage SuperNetwork EdgeWeights lt String gt ZRule lt boolean gt NumberOfRuns lt int gt SuperTree lt boolean gt ApplyRefineHeuristic lt boolean gt Input Trees Output Splits TreeSelector This method is used to select a single tree from a set of trees Usage TreeSelector Which lt int gt Input Trees Output Splits UncorrectedP This method calculates uncorrected observed P distances This is identical to the Hamming method Usage Uncorrected_P ignoregaps lt boolean gt Input Characters Output Distances UPGMA This method computes UPGMA tree 29 Usage UPGMA Input Distances Output Trees 22 Command Line Options and Mode SplitsTree4 has the following command line options 47 g lt switch gt default true GUI mode p lt String gt default HOME SplitsTree def Properties file i lt String gt default Input file x lt String gt default Execute this command at startup V lt switch gt default false show version string S lt switch gt default false silent mode d lt switch gt default false debug mode s lt switch gt default true show startup splash screen h lt switch gt default false Show usage Launching the program with option g will make the program run in non GUI command line mode first reading commands from a fi
6. already been kept Closest Tree makes the splits compatible by computing the closest tree Greedy Weakly Compatible uses a greedy approach to makes the splits weakly compatible in decreasing order of weight the algorithm adds the next split to the set of kept splits if it is weakly compatible with all splits that have already been kept Weight Threshold removes any split whose weight does not ex ceed the given threshold Pressing the Set but ton will open a histrogram and slider to set the threshold Confidence Threshold removes all splits whose confidence does not ex ceed the given threshold A confidence value usually lies in the range 0 1 and can be ob tained by bootstrapping for example Press ing the Set button will open a histrogram and slider to set the threshold Set Maximum Dimension greedily removes a subset of splits that cause boxes in the network of dimension higher than the given threshold as described in 18 None applies no filter Use the Exclude Selected Splits to remove all splits that are currently selected in the displayed network 16 Export and Export Images Dialogs 16 1 Export The Export dialog is opened using the File Export item It is used to save individual blocks of data in any of the formats described in Section 20 The dialog show two lists On the left the set of available Nexus blocks Is listed Depending on the selection of blocks made by the user on the right the set of av
7. taxon list CHARSET charset name character list 37 CHARPARTITION charpart name 1 charset name character list END 19 11 ST Assumptions Block The ST_Assumptions block controls the processing of the data along the pipeline It contains all choices made by the user that affect computations It has the following syntax BEGIN ST_ASSUMPTIONS UNALIGNTRANSFORM name parameters CHARTRANSFORM name parameters DISTTRANSFORM name parameters SPLITSTRANSFORM name parameters SPLITSPOSTPROCESS NO LEASTSQUARES FILTER GREEDYCOMPATIBLE WEAKLYCOMPATIBLE WEIGHT VALUE value CONFIDENCE VALUE valueDIMENSION VALUE value NONE EXTAXA NONE list of original taxa labels EXCHAR 1NONE list of original char positions EXCLUDE NO GAPS NO NONPARSIMONY NO CONSTANT CONSTANT number NO CODON1 NO CODON2 NO CODON3 EXTREES 1NONE list of original tree labels LAYOUTSTRATEGY STABILIZE SNOWBALL KEEP CENO AUTOLAYOUTNODELABELS UPTODATE END 20 File Formats By default SplitsTree4 reads and writes data in Nexus format The program can read and export the following additional formats FastA Phylip and ClustalW 38 Name file suffix data type new Nexus nex nxs taxa unaligned sequences aligned sequences charac ters distances quartets trees splits networks old Nexus NeX nxs aligned characters distances 25 trees FastA fa
8. 1990 N Saitou and M Nei The Neighbor Joining method a new method for reconstructing phy logenetic trees Molecular Biology and Evolution 4 406 425 1987 R R Sokal and C D Michener A statistical method for evaluating systematic relationships University of Kansas Scientific Bulletin 28 1409 1438 1958 M A Steel Recovering a tree from the leaf colorations it generates under a markov model Appl Math Lett 7 2 19 24 1994 D L Swofford G J Olsen P J Waddell and D M Hillis Chapter 11 Phylogenetic inference In D M Hillis C Moritz and B K Mable editors Molecular Systematics pages 407 514 Sinauer Associates Inc 2nd edition 1996 J D Thompson D G Higgins and T J Gibson CLUSTAL W improving the sensitivity of progressive multiple sequence alignment through sequence weighting position specific gap penalties and weight matrix choice Nucl Acids Res 22 4673 4680 1994 5l Index SplitsTree def 28 aln 39 dist 39 dst 39 fa 39 fasta 39 nex 39 nxs 39 phy 39 27 About 22 32 About command 48 All 16 allopolyploidization 9 Allow Graph Editing 29 Analysis 21 Analysis gt Bootstrap 21 31 Analysis Compute Phylogenetic Diversity 21 Analysis Conduct Phi test for Recombination 21 Analysis Configure Pipeline 5 11 21 24 Analysis Configure Recent Methods 5 11 21 23 Analysis Estimate Invariable Sites 21 Analysis gt Show Boo
9. Export A picture of the computed network can be saved using the File Export Image menu item 7 Estimating Distances Many methods in phylogenetics begin by estimating distances between the taxa This is done by taking sequences two at a time and estimating the average number of mutations that occurred on the paths from them to their most recent common ancestor If the rate of mutation was constant then this will be proportional to their divergence time SplitsTree4 provides a large number of methods for estimating distances for sequences and other types of data The UncorrectedP method computes the proportion of positions at which two sequences differ For DNA or RNA sequences there are choices over how ambiguous state codes such as W M V are handled Ignore means that these states are treated as missing states Average means that the contribution at a site is averaged over all possible resolutions of the ambiguous codes with the exception that sites having the same ambiguous code contribute zero Match looks at each possible state in each sequence counts one if the state is not a possible resolution of the ambiguous 10 code in the other sequence and normalises the count by the number of states for each ambiguous code The Distances menu lists several standard distance estimation methods Most of these have parameters that can be changed When you select the menu item the Pipeline window will open and will display a pa
10. MRJAdapter a Java package used to help construct user interfaces for the Apple Macintosh References 1 H J Bandelt and A W M Dress A canonical decomposition theory for metrics on a finite set Advances in Mathematics 92 47 105 1992 2 H J Bandelt P Forster B C Sykes and M B Richards Mitochondrial portraits of human population using median networks Genetics 141 743 753 1995 49 3 10 11 12 13 14 15 16 17 18 G S Brodal R Fagerberg A Ostlin C N S Pedersen and S S Rao Computing refined buneman trees in cubic time Lecture Notes in Computer Science 2812 259 270 2003 Springer Verlag T Bruen H Philippe and D Bryant A quick and robust statistical test to detect the presence of recombination Genetics in press 2005 D Bryant and V Moulton NeighborNet An agglomerative method for the construction of planar phylogenetic networks In R Guig and D Gusfield editors Algorithms in Bioinfor matics WABI 2002 volume LNCS 2452 pages 375 391 2002 P Buneman The recovery of trees from measures of dissimilarity In F R Hodson D G Kendall and P Tautu editors Mathematics in the Archaeological and Historical Sciences pages 387 395 Edinburgh University Press 1971 A W M Dress and D H Huson Constructing splits graphs IEEE ACM Transactions in Computational Biology and Bioinformatics 1 3 109 115 2004 R C Edgar MUSCLE multiple s
11. Tree 15 File Tools Load Trees 15 Filter Characters 18 Filter Splits 18 Filter Taxa 17 Filter Trees 18 FilteredSuperNetwork 20 41 Find Replace 16 24 31 Fit 29 fit 29 Flip 17 From Graph 25 gap 17 GapDist 41 gene content 41 GeneContent Distance 19 41 GIF 28 Go to Line 16 graphical attributes 17 Greedily Make Compatible 18 Greedily Make Weakly Compatible 18 Greedy Compatible 27 Greedy Weakly Compatible 27 Group Identical Haplotypes 15 Hamming 42 Hasegawa Kishino and Yano model 42 Help command 48 Hide All 25 Hide Incompatible Splits 22 Hide Label 23 24 hide list 25 Hide Non Selected Splits 22 Hide Selected Splits 18 22 HideSelectedSplits 12 Highlight Confidence 17 31 HKY85 19 42 How to Cite 22 How to cite 4 hybridization 9 hybridization network 9 12 42 HybridizationNetwork 21 42 import 18 Import command 48 initial calculations 19 intervals 31 Invert Selection 14 16 Jama 49 JPEG 28 JTI 45 Jukes Cantor model 42 JukesCantor 19 42 54 K2P 19 42 K3ST 19 42 Keep Only Selected Taxa 17 Kimura 2P 42 Kimura 35T 42 layout 29 least squares 26 least squares fit 30 Linux 5 6 Load 19 Load command 48 Load Multi Labeled Tree 15 Load Trees 15 LogDet 19 43 LogHamming 43 LSFit 30 Mac OS 5 MacOS 6 Main 12 Main Data 13 Main Network 13 Main Source 13 Maintain Edge Lengths 29 maintain
12. Trees Next Tree 15 Pipeline Window The Pipeline window is accessed using the Analysis Configure Pipeline item It con trols all aspects of the computational pipeline that SplitsTree4 uses It is organized into nine tabs reflecting the order of the blocks in the pipeline Pipeline Taxa Pipeline Unaligned Pipeline Characters Pipeline Distances Pipeline Quartets Pipeline Trees and Pipeline Splits 24 This is a potential source of confusion because in the main menus of the Main window we organize the methods by the type of their output rather than by the type of their input as is done here Each tab in controls how a block of the given type is processed to produce a block of a subsequent type Each of the tabs contains upto three sub tabs as mentioned below Here we now describe each of the main tabs 15 1 Taxa Tab The Pipeline Taxa tab consists of precisely one Pipeline Taxa Filter sub tab which is used to include show or exclude hide taxa from all calculations All taxa listed in the show list are included in all calculations where as all taxa listed in the hide list are removed from the data set If a set of taxa are selected in the network displayed in the Main window then these can be added to the show or hide list by pressing the appropriate From Graph button Press Show All or Hide All to show all hide all known taxa Please note that the set of shown or hidden taxa will change automatically when viewi
13. combination If the splits block contains confidence intervals for the edges for example in a confidence network then these intervals can be displayed by selecting the intervals check box Please note that any changes made only apply to the currently selected nodes or edges If there are no edges or nodes selected then changes will apply to all nodes and edges Any change made in this dialog box can be reversed using Edit Undo 18 6 Highlight Confidence Window The Highlight Confidence Window is opened using the View Highlight Confidence window item Use this window to request that edges and or edge shading is done to reflect the confidence values associated with each split 18 7 Bootstrap Window The Bootstrap window is opened using the Analysis Bootstrap item Enter the Number of Replicates and then press Run to execute 31 18 8 Message Window The Message window is opened using the Window Message Window item The program writes all messages to this window 18 9 About Window The About Window is opened using the Window About item It reports the version of the program 19 Nexus Blocks In this section we describe the Nexus format as implemented in SplitsTree4 based on the definition provided in 25 Unfortunately there exist two variants of the Nexus format which we will call old Nexus and new Nexus SplitsTree4 is based on the latter as this is what is defined in 25 Given a file formatted in old Nex
14. items e The Data Keep Only Selected Taxa item removes all taxa from the analysis whose nodes in the network are unselected All data associated with the removed taxa is removed from the original source block and all subsequent data is recomputed e The Data Exclude Selected Taxa item removes all taxa from the analysis whose nodes in the network are selected All data associated with the removed taxa is removed from the original source block and all subsequent data is recomputed e The Data Restore All Taxa item restores all taxa that where previously removed using either of the previous two menu items or by the next menu item e The Data Filter Taxa item opens the Pipeline Taxa Filter tab that can be used to interactively include or exclude taxa from the analysis e The Data Exclude Gap Sites item excludes from computation all sites in a Characters block that contain a gap or missing character in any of the sequences 17 e The Data Exclude Parsimony Uninformative Sites item excludes from computation all sites in a Characters block that are parsimony uninformative that is which are constant across all but one sequence e The Data Exclude Constant Sites item excludes from computation all sites in a Characters block that are constant across all sequences e The Data Restore All Sites item restores all sites that where excluded using the above menu items e The Data Filter Characters item opens the Pipeline Charact
15. items that determine how to compute distances trees or a network from the given data Some of the methods provided have parameters that can be set using the Analysis Configure Pipeline or Analysis Configure Recent Methods items The computed data can be viewed in text form in the Main Data tab and can be saved using the File Save As item The computed network or tree is displayed in the Main Network tab Attributes of the network can be changed by selecting nodes or branches also called edges and using the Nodes and Edges dialog which is reachable using the View Nodes and Edges item Individual blocks of data can be saved in a number of different formats using the File Export item The network displayed in the Main Network tab can be saved in a graphics format using the File Export Image item 3 Obtaining and Installing the Program SplitsTree4 is written in Java and requires a Java runtime environment version 1 4 2 or newer freely available from www java org SplitsTree4 is installed using an installer program that is freely available from www splitstree org There are four different installers targeting different operating systems splitstree_windows_4 6 exe provides an installer for Windows e splitstree_macos_4 6 sit provides an installer for Mac OS e splitstree_linux_4 6 rpm provides a RPM package for Linux e splitstree_unix_4 6 sh provides a shell installer for Linux and Unix 4 Program Overview In this sectio
16. to the current Distances block See Section 21 for a description of all available methods 15 5 Quartets Tab The Pipeline Quartets tab has only one sub tab The Pipeline Quartets Method sub tab is used to choose and configure a method to apply to the current Quartets block See Section 21 for a description of all available methods 15 6 Trees Tab The Pipeline Trees tab has three sub tabs The Pipeline Trees Method sub tab is used to choose and configure a method to apply to the current Trees block See Section 21 for a description of all available methods The Pipeline Trees Filter sub tab is used to exclude trees sites from the analysis The Pipeline Trees Select sub tab is used to highlight a set of trees in the displayed network by selecting all splits in the network that are contained in the set of chosen trees 15 7 Splits Tab The Pipeline Splits tab has two sub tabs The Pipeline Splits Method sub tab is used to choose and configure a method to apply to the current Splits block See Section 21 for a description of all available methods The Pipeline Splits Filter sub tab is used to modify the weights of splits using a least squares optimization or to exclude certain splits from the analysis Possible filters are 26 Greedy Compatible uses a greedy approach to makes the splits compatible in decreasing order of weight the algorithm adds the next split to the set of kept splits if it is compatible with all splits that have
17. 15 to produce a consensus network Usage ConsensusNetwork Threshold lt double gt EdgeWeights lt String gt Input Trees Output Splits ConsensusTree This method computes different types of consensus trees Usage ConsensusTree EdgeWeights lt String gt Method lt String gt Input Trees Output Splits ConvexHull This method computes a splits graph using the convex hull extension algorithm 7 Usage ConvexHull Weights lt boolean gt ScaleNodesMaxSize lt int gt Input Splits Output Network DNA2Splits This method converts DNA characters to splits by setting the majority state against all other states Usage DNA2Splits AddAllTrivial lt boolean gt MinSplitWeight lt int gt Input Characters Output Splits DQuartets This method compute all quartets with positive isolation index 1 Usage DQuartets threshold lt double gt Input Distances Output Quartets 40 EqualAngle This method computes a planar split network for a circular sub set of splits 7 If the RunConvexHull option is chosen then the convex hull algorithm is subsequently applied to obtain a graph for the complete set of splits This method provides a number of heuristics for obtaining a better layout 11 set DaylighIterations to 5 to apply the equal daylight heuristic set OptimizeBoxesIterations to 10 to apply the box opening heuristic and set SpringEmbedderIterations to 500 to apply a modified spring embedder Usage EqualAngle
18. 22 Data Filter Characters 18 Draw Hide Selected Splits 18 22 Data Filter Splits 18 Draw HideSelectedSplits 12 Data Filter Taxa 17 Draw NoGraph 22 Data Filter Trees 18 Draw Phylogram 22 24 Data Greedily Make Compatible 18 Draw Redraw All Splits 22 Data Greedily Make Weakly Compatible 18 Draw RootedEqualAngle 22 24 Data Keep Only Selected Taxa 17 Draw Select Characters 22 Data Load 19 Draw Select Trees 12 22 Data Restore All Sites 18 Duplicate 15 24 Data Restore All Splits 18 Data Restore All Taxa 17 Edge 23 Data Set Tree Names 18 edge 8 datatype 15 30 edge color 31 Dayhoff 45 edge font 31 DaylighIterations 41 edge width 31 default calculations 7 Edge Configure 24 Delete 25 Edge Copy Label 23 delete a node 29 Edge Edit Label 23 delete an edge 29 Edge Hide Label 24 Delete excluded command 48 Edge Show Confidence 24 diploid 9 Edge Show Id 23 Edge Show Interval 24 Disclaimer 4 Edge Show Weight 24 display of sites 26 Distances 17 19 34 Edit 16 Distances gt F81 19 Edit Label 23 Distances gt F84 19 Edit Copy 16 Distances GeneContent Distance 19 Edit Cut 16 Distances gt HKY85 19 Edit Find Replace 16 24 31 Distances JukesCantor 19 Edit Go to Line 16 Distances K2P 19 Edit Invert Selection 16 Distances gt K3ST 19 Edit Paste 16 Distances LogDet 19 Edit Preferences 16 24 28 Dista
19. IES PARTIALTREES YES NO TRANSLATE nodeLabel1 taxoni nodeLabel2 taxon2 nodeLabelN taxonN sl TREE namel treel in Newick format TREE name2 tree2 in Newick format TREE nameM treeM in Newick format END 19 7 Splits Block The splits block contains a list of splits It has the following syntax BEGIN Splits DIMENSIONS NTAX number of taxa NSPLITS number of splits FORMAT LABELS LEFT NO WEIGHTS YES NO CONF IDENCES YES NO INTERVALS YES NO a THRESHOLD non negative number PROPERTIES FIT non negative number leastsquares COMPATIBLE CYCLIC WEAKLY COMPATIBLE INCOMPATIBLE 3 CYCLE taxon_i_1 taxon_i_2 taxon_i_ntax SPLITSLABELS label_1 label_2 label_nsplits MATRIX label_1 weight_1 confidence_1 split_1 label_2 weight_2 confidence_2 split_2 35 label_nsplits weight_nsplits confidence_nsplits split_nsplits END 19 8 Network Block The Network block contains the definition of a phylogenetic network It has the following syntax BEGIN NETWORK DIMENSIONS NTAX number taxa NVERTICES number vertices NEDGES number edges DRAW ROTATE rotation 5 TRANSLATE vertex_1 taxon_1 vertex_2 taxon_2 vertex_ntax taxon_ntax A VERTICES 1 x_1 y_1 WIDTH n HEIGHT n SHAPE RECT OVAL FGC color BGC color LINE n 2 x_2 y_2 WIDTH n HEIGHT n SHAPE rect OVAL FGC color BGC color LINE n nvertices x_nvertices y_nve
20. JukesCantor method The Distances gt K2P item requests the program to compute distances using the K2P method The Distances K3ST item requests the program to compute distances using the K3ST method The Distances F81 item requests the program to compute distances using the F81 method The Distances F84 item requests the program to compute distances using the F84 method The Distances ProteinMLdist item requests the program to compute distances using the ProteinMLdist method The Distances NeiMiller item requests the program to compute distances using the NeiMiller method The Distances GeneContentDistance item requests the program to compute distances using the GeneContentDistance method Trees Menu Trees menu contains the following items 19 12 7 The The Trees gt NJ item requests the program to compute a tree using the NJ method The Trees BIONJ item requests the program to compute a tree using the BIONJ method The Trees UPGMA item requests the program to compute a tree using the UPGMA method The Trees gt BunemanTree item requests the program to compute a tree using the BunemanTree method The Trees RefinedBunemanTree item requests the program to compute a tree using the RefinedBunemanTree method The Trees TreeSelector item requests the program to compute a tree using the TreeSelector method If the Trees TreesSelector is selected then the Trees Previous Tree and Trees gt Next
21. Next Tree items can be used to move from one tree to the next in the Trees block Pressing the shift key when selecting either of these methods will move to the first tree or the last tree respectively The Trees ConsensusTree can be used to compute the majority or strict consensus tree As with distances when you select such a menu item the Pipeline window will open and will display a panel for setting the parameters of the method If you always use the same parameters for a given method or if the method has no parameters then selecting the Don t show this dialog to configure this method again will prevent the dialog from appearing again All methods can be configured using the Pipeline window accessed by Analysis Configure Pipeline or Analysis Configure Recent Methods 9 Building and Drawing Networks The Network menu provides methods for computing phylogenetic networks from character se quences distances and trees 11 Methods that compute a split network directly from character sequences provided in the Characters block are ParsimonySplits MedianNetwork SpectralSplits Note that the Me dian network method requires binary sequences However given DNA or RNA this program will detect all sites that contain precisely two character states and will build a Median network from these Two methods for computing split networks from distances provided in the Distances block are SplitDecomposition and NeighborNet If a set o
22. T NO 3 MATRIX taxonlabel1 sequence taxonlabel2 sequence taxonlabelN sequence END 19 3 Characters Block The Characters block contains aligned character sequences It has the following syntax BEGIN CHARACTERS DIMENSIONS NTAX number of taxa NCHAR number of characters FORMAT DATATYPE STANDARD DNA RNA PROTEIN RESPECTCASE MISSING symbo1 GAP symbo1 SYMBOLS symbol symbol LABELS NO LEFT TRANSPOSE NO YES INTERLEAVE NO YES TOKENS N0 33 CHARWEIGHTS wgt_1 wgt_2 wgt_nchar CHARSTATELABELS character number character name state name state name MATRIX sequence data in specified format END 19 4 Distances Block The Distances block contains a matrix of pairwise distances It has the following syntax BEGIN DISTANCES DIMENSIONS NTAX number of taxa FORMAT TRIANGLE LOWER UPPER BOTH NO DIAGONAL LABELS LEFT NO J MATRIX distance data in specified format END 19 5 Quartets Block The Quartets block contains a list of quartets It has the following syntax BEGIN Quartets DIMENSIONS NTAX number of taxa NQUARTETS number of quartets FORMAT LABELS LEFT NO WEIGHTS YES NO 5 MATRIX label1 weight1 a1 b1 c1 di labeln weightn an bn cn dn END 34 19 6 Trees Block The Trees block contains a list of phylogenetic trees It has the following syntax BEGIN Trees PROPERT
23. Tree items are used to move from one tree to the next in the Trees block Pressing the shift key when selecting either of these methods will move to the first tree or the last tree respectively The Trees ConsensusTree requests the program to compute compute the majority or strict consensus tree The Trees PhylipParsimony item requests the program to compute a tree using the PhylipParsimony method The Trees gt PhyML item requests the program to compute a tree using the PhyML method Network Menu Networks menu contains the following items The Networks NeighborNet item requests to compute a network using the NeighborNet method The Networks SplitDecomposition item requests to compute a network using the SplitDecomposition method The Networks ParsimonySplits item requests to compute a network using the ParsimonySplits method The Networks ConsensusNetwork item requests to compute a network using the ConsensusNetwork method The Networks SuperNetwork item requests to compute a network using the SuperNetwork method The Networks FilteredSuperNetwork item requests to compute a network using the FilteredSuperNetwork method 20 12 8 The 12 9 The The Networks MedianNetwork item requests to compute a network using the MedianNetwork method The Networks SpectralSplits item requests to compute a network using the SpectralSplits method The Networks HybridizationNetwork item requests to c
24. UseWeights lt boolean gt RunConvexHull lt boolean gt DaylightIterations lt int gt OptimizeBoxesIterations lt int gt SpringEmbedderlterations lt int gt Input Splits Output Network FilteredSuperNetwork This method computes a super network from partial trees using the Z closure algorithm 18 and a distortion filter 23 Usage FilteredSuperNetwork MinNumberTrees lt int gt MaxDistortionScore lt int gt EdgeWeights lt String AllTrivial lt boolean gt UseTotalScore lt boolean gt Input Trees Output Splits F81 This method calculates distances using the Felsenstein 81 model 31 Usage F81 Maximum_Likelihood lt boolean gt Estimate_Base_Frequencies lt boolean gt Base_Freqs lt N double1 double2 doubleN gt Normalize lt boolean gt Input Characters Output Distances F84 This method Calculates distances using the Felsenstein 84 model 31 Usage F84 Maximum_Likelihood lt boolean gt Estimate_Base_Frequencies lt boolean gt Normalize lt boolean gt TRatio lt double gt A lt double gt C lt double gt G lt double gt T_U lt double gt Input Characters Output Distances GapDist This method calculates the gap distance from a set of sequences Usage GapDist Input Characters Output Distances GeneContentDistance This method compute distances based on gene content 19 Al Usage GeneContentDistance UseMLDistance lt boolean gt Input Characters Output Distances Hamming
25. User Manual for Splits Tree4 V4 6 Daniel H Huson and David Bryant August 4 2006 written by Daniel H Huson and David Bryant 2005 with contributions from se eggs Miguel Jette Tobias Kl pper MESS el www splitstree org Contents Contents 1 Introduction 2 Getting Started 3 Obtaining and Installing the Program 4 Program Overview 5 Splits Trees and Networks 6 Opening Reading and Writing Files 7 Estimating Distances 10 10 8 Building and Processing Trees 9 Building and Drawing Networks 10 Main Window Wh a nd TaD a oa ets 2 ae hea Ae ewe Bs ee cs A le en de we a a G 10 2 Data Tab occiso sd sra aa EE ER ee Seed Ge k TUS pomeo TESSA o e ir A ee hee ee A bee MA Tool Bartolo ec eee es by AA a Se eas GOR Se pose ee Sees 11 Graphical Interaction with the Network 12 Main Menus UML Vie Mei oe oR ee a a ee Be rere LA Bee 122 Bi Memi as ee os NN 12 View Menn oe ete ee OR ae Be Eee Oe he ee Sey Oe Rae ee ad ZA Date Meme osas Saheb aan eb oe a Ra os a Dean SD e o pene a ee as aoe Ses 126 Trees Mem 2 o ee a aaa A oe RRA ae Ew 12 7 Network Menu 24 0 24 3 0654 rar raras BEY Roe Re was 4 Toe Pers Meme eo a ere Se sed a Ba Wee ai a ee Se Ae eee 129 Draw Men ooo ocaso ee ea a Pe dae eee T210Window Menu cco cacag bee ee RSS RAE Re ESE A EC Methods s i on oe gel ke A we a a a Bo ee we aie 13 Popup Menus 14 Tool Bar 15 Pipeline Window 19 1 Tara VES e lk ek eee Pew eR ek aed bow be Py
26. a The aim of SplitsTree4 is to provide a frame work for computing phylogenetic networks As the name of the program suggests it is based on the fundamental mathematical concept of a split For example if we are given an alignment of binary sequences a 010011010110 b 100001011110 c 011001101110 d 010001101111 then each non constant column in the alignment defines a split of the taxon set consisting of those taxa with the value 0 and those with the value 1 For example the first column partitions the taxa into two sets a c d and b and thus gives rise to the split Tp while the fourth column does not define a split because the characters are constant Mathematically for a set of X taxa any phylogenetic tree T defines a set of such splits called the split encoding X T of T as follows deletion of any branch also called edge ein the tree produces two subtrees T4 and Tg say and they give rise to a split S 4 Z consisting of the set A of all taxa contained in T4 and the set B of all taxa contained in Tg In the literature a split 4 is sometimes denoted by A B or A B For example consider the tree T displayed in Figure 1 Its split encoding X T contains 5 trivial splits that separate a single taxon from all other taxa and 2 non trivial splits that contain at least two taxa in both parts The trivial splits are fo 0 eh a a b c d e a c d e a b d e a b c ey a b c d and the non trivial
27. a eee e ee od 15 2 Unalgned Ta o ecca a nioi radar eS REE Re ee we 8 4 ce CNR cis eo i ee de a wad ee ed et ee Ry hn a ee 194 Distances Tab s pescia eo a he ee ee a bee d a D Api 2a ee eo lo Guarese TAG cc eae a a a e A Eo ee ee a we 15 0 Tress Tab cc a cede hee eAGbe eee eee be hha A EER ee 11 11 12 13 13 13 13 13 14 14 16 16 17 19 19 20 21 21 22 23 23 24 15 7 Splits Tab 254 16 Export and Export Images Dialogs 16 1 Export 4 4 4 16 2 Export Image 17 Preferences Window 18 Additional Windows and Dialog 18 1 Open File 18 2 Choose Datatype 13 3 Bave AS sacks 4 da 18 4 Find Replace Window 18 5 Node and Edge Window 5 55454 bbe Re REE Re ER ee eR ee es 186 Meblight Confidence Winddw gt e se 4 6404 ee a ee ES ES 18 7 Bootstrap Window 18 8 Message Window 18 9 About Window 19 Nexus Blocks 19 1 Taxa Block 2 4 4 19 2 Unaligned Block 19 3 Characters Block 19 4 Distances Block 19 5 Quartets Block 19 6 Trees Block 19 7 Splits Block 19 8 Network Block 19 9 Bootstrap Block 19 10Sets Block 19 11ST Assumptions Block 20 File Formats 21 All Methods 27 27 28 28 30 30 30 30 31 31 31 31 32 32 32 32 33 33 34 34 35 35 36 37 37 38 38 39 22 Command Line Options and Mode 47 23 Examples 49 24 Acknowledgments 49 References 49 Index 52 1 Introduction Dis
28. ailable export formats is listed Most of the dependences between blocks and formats should be apparent One interesting feature is the following If one has enabled graph editing using the Allow Graph Editing option in the Preferences General tab and has interactively constructed a phylogenetic tree then this tree can 27 be saved in Newick format Hence SplitsTree4 can be used for editing trees 16 2 Export Image The Export Image dialog is opened using the File Export Image item This dialog is used to save a picture of the current network in a number of different formats The following graphics formats are supported JPEG Joint Photographic Experts Group e GIF Graphics Interchange Format e EPS Encapsulated PostScript SVG Scalable Vector Graphics e PNG Portable Network Graphics e BMP Bitmap There are two radio buttons the Save whole image to save the whole image and the Save visible image to save only the part of the image that is currently visible in the main viewer If the chosen format is EPS then selecting the Convert text to graphics check box will request the program to render all text as graphics rather than fonts Pressing the apply button will open a standard file save dialog to determine where to save the graphics file 17 Preferences Window The Preferences window is opened using the Edit Preferences item This window controls program preferences Many of th
29. claimer This software is provided AS IS without warranty of any kind This is develop mental code and we make no pretension as to it being bug free and totally reliable Use at your own risk We will accept no liability for any damages incurred through the use of this software Use of the Splits Tree4 is free however the program is not open source Type setting conventions In this manual we use e g Network NeighborNet to indicate the NeighborNet menu item in the Network menu We use e g Main Source to indicate the Source tab in the Main window How to cite If you publish results obtained in part by using SplitsTree4 then we require that you acknowledge this by citing the program as follows e D H Huson and D Bryant Application of Phylogenetic Networks in Evolutionary Stud ies Molecular Biology and Evolution 23 2 254 267 2006 software available from www splitstree org or e D H Huson Splits Tree A program for analyzing and visualizing evolutionary data Bioinfor matics 14 10 68 73 1998 Evolutionary relationships are usually represented using phylogenetic trees based on a model of evolution dominated by mutations and speciation events More realistic models must also account for gene genesis loss and duplication events hybridization horizontal gene transfer or recombina tion Here phylogenetic networks have a role to play Moreover network methods also provide a value tool for phylogenetic inference even wh
30. d to perform certain tasks The Main window has three tabbed panes e the Main Network tab is used to display the computed tree or network e the Main Data tab can be used to inspect all data associated with a given document in text format and e the Main Source tab can be used to inspect or edit the source data from which all other data is derived We now discuss each separately 12 10 1 Network Tab The Main Network tab is used to display the computed tree or network A status line of text along the bottom of the network pane gives a summary of the data such as number of taxa length of sequences etc and a summary of the methods used to compute the network Optionally a scale bar can be displayed in the upper left corner of the Network tab to indicate the scale of branches The scale bar is can be turned on or of using the Preferences General tab The scale bar can be configured using the Preferences Status Line tab 10 2 Data Tab The Main Data tab provides a textual display of the data associated with the given document in the program s native Nexus format organized in a linear list of items that can be either collapsed or expanded This view of the data is read only 10 3 Source Tab The Main Source tab provides an editable view of the source data associated with the given view This view provides three different functionalities namely e data can be entered by hand or by copy and paste in any of the file formats
31. dopsin 45 RootedEqualAngle 22 24 46 Rotate Left 17 24 Rotate Right 17 24 Run 31 RunConvexHull 41 RYSplits 46 sampling error 4 Save 7 10 15 Save As 5 7 10 15 24 30 Save command 48 Save visible image 28 Save whole image 28 scale bar 29 Select All 16 Select Characters 22 Select Trees 12 22 sequence alignment 39 43 44 Set Maximum Dimension 27 Set Tree Names 18 56 Sets 37 Show All 25 Show Bootstrap Network 21 Show Confidence 24 Show Confidence Network 21 Show cycle command 48 Show Id 23 Show Interval 24 show list 25 Show Name 23 Show Scale Bar 29 Show Weight 24 source block 7 source text area 18 SpectralSplits 21 46 split 8 split confidences 31 split decomposition 47 split encoding 8 split ids 31 split network 9 split weights 31 Split Selection Mode 14 SplitDecomposition 20 47 Splits 17 splits 35 SplitsTree 48 splitstree_linux_4 6 rpm 5 splitstree_macos_4 6 sit 5 splitstree_unix_4 6 sh 5 splitstree_windows_4 6 exe 5 SpringEmbedderlterations 41 ST_Assumptions 38 status line 29 super network 12 super network 41 47 SuperNetwork 20 47 SVG 28 synchronized 7 systematic error 4 Taxa 32 TAXLABELS 33 taxon ids 31 taxon names 31 Taxon Sets 16 tool bar 13 24 Tools 15 tree 7 tree names 18 Trees 19 35 Trees gt BIONJ 20 Trees gt BunemanTree 20 Trees ConsensusTree 11 20 Trees g
32. e preferences are persistent that is they remain effective even after closing and re opening the program To remember these choices SplitsTree4 creates and maintains a properties file which is placed in the users home directory and is called something like Splits Tree def It has five tabs The Preferences General tab controls general aspects of the program 28 Allow Graph Editing if set the user can add or delete nodes and edges from dis played network To create a node control double click on the canvas To create an edge between two nodes control click on a node and then drag create an edge If dragging ends on a second node then this is connected to the first otherwise a new node is created To delete a node or delete an edge select it and press the delete key Maintain Edge Lengths Usually when interacting with a displayed network we want the network to maintain edge lengths Show Scale Bar The program uses a small scale bar in the top left hand corner of the Main Network tab to indicate the scale of the network use Split Selection Mode When this feature is on clicking on an edge will select all edges that correspond to the same split Any choices made here can either be applied to the current document or can be made the default for all subsequently opened documents The Preferences Defaults tab controls the default sizes shapes colors and fonts for nodes and edges see also Nodes and Edges The Prefer
33. edge lengths 29 maximum likelihood 45 maximum parsimony 44 median network 43 MedianNetwork 21 43 merge a set of trees 15 Message 32 Message Window 23 24 32 missing character 17 MRJAdapter 49 mtMAM 45 mtREV24 45 multi threading 6 multiple documents 6 Muscle 43 Neighbor Net 44 NeighborJoining 23 NeighborNet 20 44 NeiMiller 19 43 Network 36 network 8 Networks 20 Networks ConsensusNetwork 20 Networks FilteredSuperNetwork 20 Networks HybridizationNetwork 21 Networks MedianNetwork 21 Networks NeighborNet 20 Networks ParsimonySplits 20 Networks RecombinationNetwork 21 Networks SpectralSplits 21 Networks SplitDecomposition 20 Networks SuperNetwork 20 New 14 new Nexus 32 New taxa set 16 Next Tree 11 20 24 Nexus 32 Nexus Syntax 22 NJ 20 43 Noalign 44 Node 23 node color 31 node font 31 node shape 31 node size 31 Node Configure 23 Node Copy Label 23 Node Edit Label 23 Node Exclude Selected Taxa 23 Node Hide Label 23 Node Show Id 23 Node Show Name 23 Nodes and Edges 5 14 17 29 31 Nodes and Edges Edges 31 Nodes and Edges Nodes 31 NoGraph 22 44 non trivial splits 8 None 27 NoSplits 44 Number of Replicates 31 old Nexus 32 Open 5 10 15 19 24 30 Open File 30 Open Recent 10 15 OptimizeBoxeslterations 41 p 23 parallel branches 9 parallel edges 9 par
34. en retic ulation events do not play an important role The combined effect of sampling error and systematic error makes phylogenetics an uncertain science and network methods provide tools for representing and quantifying this uncertainty The aim of SplitsTree4 is to provide a framework for evolutionary analysis using both trees and networks The program takes as input a set of taxa represented by characters that is aligned sequences distances quartets trees or splits and produces as output trees or networks using a number of different methods This document provides both an introduction and a reference manual for SplitsTree4 2 Getting Started This section describes how to get started First download an installer for the program from from www splitstree org see Section 3 for details Use the File Open menu item to open a file containing data such as character sequences distances or trees The file must be in one of the following formats Nexus ClustalW Phylip FastA or Newick Alternatively select the Main Source tab of the Main window and enter data in one of these formats by hand Example files are provided with the program They are contained in the examples sub directory of the installation directory The precise location of the installation directory depends upon your operating system Use the different menu items to determine which methods are applied to the input data The Distances Trees and Network menus contain
35. ences Layout tab controls aspects of the layout of trees and split networks Recompute The taxon layout is recomputed each time the Splits block is modified Stabilize Use the union of the set of current splits and of the previ ously computed splits to compute a taxon layout This is useful when one wants to compare two different networks computed on the same taxon set by switching back and forth between the two computations the program will find a joint taxon layout for both graphs if one exists In this case both graphs will be laid out in such a way that in both graphs the taxa appear in approximately the same region of the canvas Snowball Similar to the previous method this method uses all splits ever computed in a given dataset to produce a taxon layout This doesn t work very well Use this layout Use the provided taxon layout to draw the network The Preferences Toolbar tab allows the user to interactively configure the tool bar The Preferences Status Line tab allows the user to configure the status line displayed along the bottom of the Main Network tab Here two items are of particular interest If the displayed graph was computed either by the BunemanTree or SplitDecomposition method then the pair wise distances in the graph may under estimate the true distances in the given Distances block The difference between the two can be expressed in terms of the fit value which is activated using the Fit check box which is de
36. equence alignment with high accuracy and high throughput Nucleic Acids Research 32 5 1792 97 2004 D P Faith Conservation evaluation and phylogenetic diversity Biol Conserv 61 1 10 1992 J Felsenstein PHYLIP phylogeny inference package version 3 6 Distributed by the author Department of Genome Sciences University of Washington Seattle 2004 P Gambette and D H Huson Improved layout of phylogenetic networks Submitted 2005 O Gascuel BIONJ An improved version of the NJ algorithm based on a simple model of sequence data Mol Biol Evol 14 685 695 1997 S Guindon and O Gascuel A simple fast and accurate algorithm to estimate large phylo genies by maximum likelihood Syst Biol 52 5 696 704 2003 M D Hendy and D Penny Spectral analysis of phylogentic data Journal of Classification 10 5 24 1993 B Holland and V Moulton Consensus networks A method for visualizing incompatibilities in collections of trees In G Benson and R Page editors Proceedings of Workshop on Algorithms in Bioinformatics volume 2812 of LNBI pages 165 176 Springer 2003 D H Huson SplitsTree A program for analyzing and visualizing evolutionary data Bioin formatics 14 10 68 73 1998 D H Huson and D Bryant Application of phylogenetic networks in evolutionary stud ies Molecular Biology and Evolution 23 2 254 267 2006 Software available from www splitstree org D H Huson T Dezulian T Kloepp
37. er and M A Steel Phylogenetic super networks from partial trees IEEE ACM Transactions in Computational Biology and Bioinformatics 1 4 151 158 2004 50 19 D H Huson and M Steel Phylogenetic trees based on gene content 20 13 2044 9 2004 20 D H Huson Introduction to phylogenetic networks Tutorial presented at ISMB 2005 21 D H Huson T Kloepper P J Lockhart and M A Steel Reconstruction of reticulate net 22 23 24 25 26 27 28 29 30 31 works from gene trees In Proceedings of the Ninth International Conference on Research in Computational Molecular Biology RECOMB 2005 D H Huson and T H Kloepper Computing recombination networks from binary sequences To appear in ECCB 2005 D H Huson M A Steel and J Whitfield Reducing distortion in phylogenetic networks To appear in WABI 2006 2006 P J Lockhart M A Steel and D H Huson Invariable site models and their uses in phylogeny reconstruction Syst Biol 49 2 225 232 2000 D R Maddison D L Swofford and W P Maddison NEXUS an extendible file format for systematic information System Bio 46 4 590 621 1997 E Mossel and M Steel A phase transition for a random cluster model on phylogenetic trees Submitted 2003 M Nei and J C Miller A simple method for estimating average number of nucleotide substi tutions within and between populations from restriction data Genetics 125 873 879
38. ers Filter tab that can be used to interactively include or exclude sites from the analysis e The Data Greedily Make Compatible item uses a greedy approach to makes the splits in the Splits block compatible in decreasing order of weight the algorithm adds the next split to the set of kept splits if it is compatible with all splits that have already been kept e The Data Greedily Make Weakly Compatible item uses a greedy approach to makes the splits in the Splits block weakly compatible in decreasing order of weight the algorithm adds the next split to the set of kept splits if it is weakly compatible with all splits that have already been kept e The Data Exclude Selected Splits item removes all splits from the analysis whose edges in the network are selected The Splits and Network block are modified accordingly See also Draw Hide Selected Splits e The Data Restore All Splits item restores all splits that were excluded using the previous menu item e The Data Filter Splits item opens the Pipeline Splits Filter tab that can be used to interactively include or exclude splits from the analysis e The Data Filter Trees item opens the Pipeline Tree Filter tab that can be used to interactively include or exclude trees from the analysis e The Data Set Tree Names item opens a dialog that allows one to set the tree names In the Main Source tab the Data contains three commands for processing the text contained in the so
39. f phylogenetic trees in the Trees block all contain the full set of taxa listed in the Taxa block then the ConsensusNetwork method can be applied to compute a consensus network If however the Trees block contains partial trees that is trees that do not necessarily all involve identical sets of taxa then the SuperNetwork or FilteredSuperNetwork method can be used to compute a super network The Networks menu also provides two methods for computing reticulate networks If the Trees block contains more than one tree then the HybridizationNetwork method can be used to compute a hybridization network for the given data set More precisely the HybridizationNetwork requires that the Trees and Splits blocks are present and non empty If the Characters block contains binary sequence data then the RecombinationNetwork method can be used to compute a recombination network The Draw menu determines which algorithm is used to construct the final visualization of the tree or network Existing methods are EqualAngle RootedEqualAngle Phylogram and ReticulateNetwork Additionally the Draw HideSelectedSplits can be used to remove se lected splits from the network and the Draw Select Trees can be used to highlight different trees contained in a split network 10 Main Window With SplitsTree4 multiple documents can be opened and worked on simultaneously Each docu ment is represented by a Main window Additional windows or dialogs are sometimes opene
40. fasta unaligned sequences or aligned characters Phylip phy dst dist aligned characters or dis 10 tances ClustalW aln aligned characters 32 21 All Methods Here we list all methods supported by SplitsTree4 We describe the usage and list the input and output Nexus blocks Binary2Splits This method converts binary characters to splits Usage Binary2Splits AddAllTrivial lt boolean gt MinSplitWeight lt int gt Input Characters Output Splits BioNJ This method computes the Bio NJ tree 12 Usage BioNJ Input Distances Output Trees BunemanQuartets This method computes all quartets with positive Buneman index 6 Usage BunemanQuartets threshold lt double gt Input Distances Output Quartets BunemanTree This method computes the Buneman tree 6 Usage BunemanTree Input Distances Output Splits ClustalW This method externally runs the ClustalW sequence alignment program 32 39 Usage ClustalW GapOpen lt int gt GapExtension lt double gt WeightMatrix lt java lang String gt OptionalParameter lt java lang String gt PathToCommand lt java lang String gt Input Unaligned Output Characters Coalescent This method transforms a set of quartets to a set of splits representing a binary phylogenetic tree by applying the coalescent method described in 26 Usage Coalescent Input Quartets Output Splits ConsensusNetwork This method computes the consensus splits of trees 2
41. fined as the sum of all pairwise distances in the graph divided by the sum of all pairwise distances in the given matrix times 100 This is not applicable to other tree or network building methods 29 For other methods please use the LSFit item which computes the least squares fit between the pairwise distances in the graph and the pairwise distances in the matrix 18 Additional Windows and Dialog Here we list all other windows 18 1 Open File The Open File dialog is opened using the File Open item Use it to open any file containing phylogenetic data in one of the formats described in Section 20 If an error is encountered then the file is opened in the MainSource tab If possible the offending line is highlighted 18 2 Choose Datatype When opening a file containing character sequences or importing sequences from the Main Source tab the program must know whether the sequences are to be interpreted as DNA RNA protein or standard 0 1 data In Nexus files the datatype is explicitly given In other file formats this is usually not the case The program employs a simple heuristic to guess the datatype If this fails e g if all character states are A then the program will display a Choose Datatype dialog and prompt the user to specify the datatype The choices are e dna e rna e protein standard which means 0 1 data and e unknown which the program cannot deal with 18 3 Save As The Save A
42. g graph is a tree In general however this will not be possible and in a split network usually a whole band of parallel branches also called parallel edges is required to represent a single split A phylogenetic tree is therefore a special case of a split network In SplitsTree4 if you click on any branch in the representation of a split network then all branches corresponding to the same split will be highlighted If one were to delete all branches corresponding to a given split S then the remaining graph will consist of precisely two components G4 and Gp and as above the we have S 4 where A is the set of all taxa contained in Gx and B is the set of all taxa contained in Gp There is an important difference between phylogenetic trees and more general split networks Any rooted tree has a direct interpretation in evolutionary terms the leaves represent taxa and the internal nodes represent speciation events In a possibly rooted split network the internal nodes do not have such a direct interpretation Instead split networks must be viewed on a more abstract level as networks giving a visual representation of incompatible signals that is showing how tree like or certain parts of a phylogeny are A reticulate network is used to describe reticulate evolution and there are two types of such networks one is the hybridization network and the other is the recombination network A hybridization network describes evolution in
43. g the Main Source tab The program is designed to operate in two differents modes in a GUI mode the program provides a GUI for the user to interact with the program In command line mode the program reads commands from a file or from standard input and writes output to files or to standard output 5 Splits Trees and Networks Here we give a brief introduction to some of the concepts from phylogenetics that the program is based on Evolutionary relationships between taxa are usually represented using a phylogenetic tree Such trees are often computed from molecular sequence data either directly using methods such as parsimony or indirectly by first computing a distance matrix and then applying a method such as Neighbor Joining Such approaches implicitly or explicitly model the evolution of a single gene under the assumption that the process is dominated by two types of events mutations and speciation events Under more complex models of evolution i e involving gene loss and duplication or hybridization horizontal gene transfer or recombination a single phylogenetic tree will often not be an appropriate repre sentation of the phylogentic history or of the different incompatible phylogenetic signals Also the presence of noise in a data set or uncertainty due to inadequacies of the underlying model up on which a tree inference is based may also make it necessary to use a more general graph that is a network to represent the dat
44. iew Reset item resets the layout of the network if used while in the Main Network tab and it clears the text area while in the Main Source tab e The View Zoom In item is used to zoom into the network 16 e The View Zoom Out item is used to zoom out from the network e The View Rotate Left and View Rotate Right items are used to rotate the network e The View gt Flip item changes the layout of the network to it s mirror image e The View Nodes and Edges item opens the Nodes and Edges window that is used to modify the graphical attributes of the nodes and edges of the network e The View Highlight Confidence item opens the Confidence window that can be used to control how confidence values for splits or edges are highlighted in the network e The View Auto Layout Node Labels check box item controls whether node labels are automatically positioned or whether the user can interactively reposition them In the Main Data tab the View menu contains the following items e The View Expand All and View gt Collapse All items expand and collapse all nodes in the displayed data tree e The View Characters View Distances and View Splits submenus control the format used to write the Characters Distances and Splits blocks respectively In the Main Source tab the View menu only contains a View Reset item which is used to clear the source text area 12 4 Data Menu The Data menu contains the following
45. ile file REPLACE YES NO TEXTASSHAPES YES NO TITLE title export graphics in specified format default is EPS UPDATE rerun computations to bring data up to date BOOTSTRAP RUNS number of runs perform bootstrapping on character data 48 DELETEEXCLUDED delete all sites from characters block that are currently excluded ASSUME assumption set an assumption using any statement defined in the ST_ASSUMPTIONS block SHOW DATA list of blocks show the named data blocks CYCLE KEEP cycle set the graph layout cycle to KEEP or to a given cycle HELP show this info HELP DATA list of blocks show syntax of named blocks HELP TRANSFORM transform show usage of a specific data transformation VERSION report version ABOUT show info on version and authors QUIT exit program To begin or end a multi line input enter a backslash 23 Examples Example files are provided with the program They are contained in the examples sub directory of the installation directory The precise location of the installation directory depends upon your operating system 24 Acknowledgments We would like to thank Barry G Hall Pete Lockhart David Morrison and Mike Steel for many helpful comments This product includes software developed by the Apache Software Foundation http www apache org namely the batik library for generating image files It also contains Jama a Java matrix package http math nist gov javanumerics jama and
46. individual edges deselect the Split Selection Mode check box in the Preferences General tab e Select a node by clicking on it Click outside of the network and drag a rectangle to select several nodes at once Hold the shift key and click to add or remove further nodes from the set selected e Click on a text label to edit it e Double click on an edge to edit its label e By default node labels are automatically positioned and cannot be moved To allow inter active positioning of node labels deselect the View Auto Layout Node Labels check box e Many aspects of the visual representation of nodes and edges can be modified using the Node and Edge Window which is opened using the View Nodes and Edges menu item 12 Main Menus We now discuss all menus of the Main window Some of the menus differ depending upon which tab is selected in the Main window 12 1 File Menu The File menu contains the usual file related items e The File New item opens a new Main window and selects its Main Source tab 14 e The File Open item provides an Open File dialog to open a file containing input data in one of the supported formats If the file contains character sequences then the program must know whether the sequences are DNA RNA protein or standard 0 1 data In Nexus files the datatype is explicitly given In other file formats this not always the case If the program cannot guess the data type e g if all character s
47. labels the selected edge by the confidence of the corresponding split e The Show Interval labels the selected edge by the confidence interval of the corresponding split e The Hide Label hides the label of the selected edge e The Configure opens the Nodes and Edges Edge tab of the Nodes and Edges window If the mouse is clicked on the drawable region of the window but not on any node or edge then the Window popup menu will open which has the following items e The Zoom In item is a short cut to the View Zoom In menu item e The Zoom Out item is a short cut to the View Zoom Out menu item e The Reset item is a short cut to the View Reset menu item e The Reset Label Positions item resets all node labels to their default positions e The Restore All Taxa item restores all excluded taxa e The Restore All Splits item restores all excluded splits 14 Tool Bar For easier access to frequently used options and methods SplitsTree4 provides a tool bar at the top of the Main window The tool bar can be configured using the Preferences Toolbar tab By de fault the tool bar contains the following items File Open File Duplicate File Save As File Print File Export Image Edit Find Replace View Reset View Zoom In View Zoom Out View Rotate Right View Rotate Left Edit Preferences Window Message Window Draw EqualAngle Draw RootedEqualAngle Draw Phylogram Trees Previous Tree and
48. lable upon publication of the paper describing this new method The File Tools Concatenate Sequences item can be used to concatenate sequences from different files into one document It requires that each of the input files uses exactly the same set of taxon labels The File Tools Group Identical Haplotypes tools is used to detect multiple identical sequences or taxa These are recognised from the Characters or if there is no Characters block from the distances block Only selected sites are used to test if two taxa are distinguishable A new document is created identical taxa are collapsed into a single taxa with label TYPEx x ranging from 1 2 3 and the original taxa stored in the info field of the Taxa block Note that files with hundreds or thousands identical sequences can cause Splitstree to stall One solution is to cancel the computations after data is read in then using this tool to create a new smaller document containing only distinct sequences e The File Print item is used to print the current network e The File Quit item quits the program after asking whether to save unsaved changes 15 12 2 Edit Menu The Edit menu contains the usual edit related items The Edit gt Undo item is used to undo text editing interactive network manipulation and any item chosen from the View menu The Edit Redo item is used to redo text editing interactive network manipulation and any item chosen from the View men
49. le supplied with the i option if any then executing any command given with the x option and then finally reading additional commands from standard input Please be aware that the command line version of the program uses the same properties file as the interactive version So any a preferences set using the interactive version of the program will also apply to the command line version of the program It this is not desired then please use the p option to supply a different properties file Commands provided to the program from within a file must be grouped together ina SplitsTree block so begin SplitsTree commands end Here we list all commands known to SplitsTree4 EXECUTE FILE file open and execute a file in Nexus format OPEN FILE file open but don t execute a file in Nexus format IMPORT FILE file DATATYPE PROTEIN RNA DNA STANDARD UNKNOWN open but don t execute a fi old Nexus format LOAD FILE file open or import a file LOAD TREEFILES file1 filen load trees from one or more files LOAD CHARFILES file1 filen concatenate sequences from one or more files LOAD FILE file open or import a file SAVE FILE file REPLACE YES NO APPEND YES NO DATA ALL list of blocks save all data or named blocks to a file in Nexus format EXPORT FILE file FORMAT format REPLACE YES NO APPEND YES NO DATA list of blocks export data in the named formatAA EXPORTGRAPHICS format EPS PNG GIF JPG SVG f
50. lt double gt Input Characters Output Distances 42 LogDet This method Calculates the LogDet distance 30 Usage LogDet Impute_Gaps lt boolean gt Input Characters Output Distances LogHamming This method calculates distances using the log Hamming distance Usage LogHamming ignoregaps lt boolean gt Input Characters Output Distances MedianNetwork This method computes an unreduced median network 2 It uses all sites in the character alignment that contain exactly two different states and no gaps or missing states If UseRYAlphabet is selected then DNA and RNA sequences are translated using R A G and Y C T U If UseRelaxedSupport is selected a character need only be constant on one side of a split to count toward the support of the split Usage MedianNetwork AddA11Trivial lt boolean gt MinimalSupport lt int gt UseRYAlphabet lt boolean gt LabelEdges lt boolean gt UseRelaxedSupport lt boolean gt Input Characters Output Splits Muscle This method externally runs the Muscle sequence alignment program 8 Usage Muscle Maxiters lt int gt ClusterMethod_1 lt String gt ClusterMethod_2 lt String gt DistanceMeasure_1 lt String gt DistanceMeasure_2 lt String gt ObjectiveScore lt String gt LogFile lt boolean gt LogFileName lt String gt OptionalParameter lt String gt PathToCommand lt String gt Input Unaligned Output Characters NeiMiller This method computes distances f
51. n we give an overview over the main design goals and features of this program Basic knowledge of the underlying design of the program should make it easier to use the program SplitsTree4 is written in the programming language Java The advantages of this is that we can provide versions that run under the Linux MacOS Windows and Unix operating systems Addi tionally this makes it possible for the program to support plug ins that can add new functionality to the program such as new methods and import or export modules A potential draw back is that an algorithm implemented in Java will generally run slower than the same algorithm implemented in C or C Earlier versions 1 3 of the program 16 were written in C and only contain a small part of what is now available with SplitsTree4 SplitsTree4 uses multi threading and supports multiple documents This means that that you can work on more than one data set simultaneously in different windows and run multiple calculations simultaneously making use of multiple processors when available A document consists of an individual data set and possesses its own Main window The document is discarded when its window is closed The program is based on the Nexus format as introduced in 25 and the data associated with a document is organized into blocks and each such block of data is represented by a corresponding block in the Nexus format The blocks are e Taxa the names of all
52. n use different menu items to determine how the program will compute data along the pipeline until a Network block has been computed and an image of the network has been drawn in the Main window The program is designed to keep all blocks in the pipeline synchronized by enforcing that the different blocks only contain data that has been computed via the pipeline For example if you attempt to load data from a file in Nexus format that contains a Taxa and two or more other blocks e g a Characters block or Distances block then the program will request you to choose which of the two latter blocks you want to keep This does not apply if the file was created by SplitsTree4 using the File Save or File Save As command in which case the blocks in the file are synchronized and so none must be discarded and no computations are necessary Once a source block has been provided the program will proceed to perform a chain of default calculations which differ depending on the type of the source block In the case of a Characters block the program will use UncorrectedP NeighborNet and then EqualAngle to compute a network for the data In the case of a Distances block NeighborNet and EqualAngle are used In the case of a Trees block the first tree in the block is displayed using the EqualAngle algorithm The data contained in the different blocks of the pipeline is displayed in the Main Data tab The data contained in the source block is edited usin
53. nces NeiMiller 19 Edit gt Redo 16 Distances ProteinMLdist 19 Edit Select All 16 Distances UncorrectedP 19 Edit Taxon Sets 16 distortion filter 41 Edit TaxonSets All 16 DNA2Splits 40 Edit TaxonSets Clear All Taxa Sets 16 document 6 Edit TaxonSets New taxa set 16 Don t show this dialog for this method again 23 Edit Undo 16 31 DQuartets 40 editing trees 28 Draw 21 Enter a Command 22 Draw ConvexHull 22 EPS 28 Draw EqualAngle 22 24 EPS command 48 equal daylight 41 53 EqualAngle 22 24 41 Estimate Invariable Sites 21 examples 5 49 Exclude Constant Sites 18 Exclude Gap Sites 17 Exclude Parsimony Uninformative Sites 18 Exclude Selected Splits 18 22 27 Exclude Selected Taxa 17 23 Execute 18 execute 18 Expand All 17 Export 5 10 15 27 Export command 48 Export Image 5 10 15 24 28 F81 19 41 F84 19 41 FastA 38 39 Felsenstein 81 41 Felsenstein 84 41 File 14 File command 48 File Close 15 File Duplicate 15 24 File gt Export 5 10 15 27 File Export Image 5 10 15 24 28 File gt New 14 File Open 5 10 15 19 24 30 File Open Recent 10 15 File Print 15 24 File gt Quit 15 File Replace 15 File Save 7 10 15 File Save As 5 7 10 15 24 30 File Tools 15 File Tools Concatenate Sequences 15 File Tools Group Identical Haplotypes 15 File Tools Load Multi Labeled
54. nel for setting the parameters of the method If you always use the same parameters for a given method or if the method has no parameters then selecting the Don t show this dialog to configure this method again will prevent the dialog from appearing again All meth ods can be configured using the Pipeline window accessed by Analysis Configure Pipeline or Analysis Configure Recent Methods 8 Building and Processing Trees The Tree menu provides a number of methods for constructing trees both from distance and character data The following distance based methods are available NJ BioNJ UPGMA Buneman and Refined Buneman The program provides front ends for several tree based programs currently PhyML and Phylip SplitsTree4 will pass these programs the current sequences and place any trees produced in the SplitsTree4 Trees block Our distribution of SplitsTree4 does not provide any external programs so you must install them separately In the dialog that is associated with such a method you must specify the location of the external application e g using the PhyML Path text field Once entered the program will remember this entry as the default value SplitsTree4 provides a number of methods for processing a collection of trees The pmenu itemTreesTreeSelector menu contains items to select individual trees or to compute a consensus of trees If the Trees TreesSelector is selected then the Trees Previous Tree and Trees
55. ng different partial trees to reflect the set of taxa contained in the current tree 15 2 Unaligned Tab The Pipeline Unaligned tab consists of precisely one sub tab The Pipeline Unaligned Method sub tab is used to choose and configure a method to apply to the current Unaligned block Currently the program provides three methods e The NoAlign item simply adds gaps to the ends of sequences to make the all have the same length e The ClustalW item can be used to call the program ClustalW externally to compute an alignment of the sequences e The Muscle item can be used to call the program Muscle externally to compute an alignment of the sequences 15 3 Characters Tab The Pipeline Characters tab has three sub tabs The Pipeline Characters Method sub tab is used to choose and configure a method to apply to the current Characters block See Section 21 for a description of all available methods The Pipeline Characters Filter sub tab is used to exclude certain sites from the analysis By default the sites remain present in the Characters block and are simply masked during calculations To remove the sites completely press the Delete button 25 The Pipeline Characters Select sub tab is used to select certain sites in the Characters block for display of sites in the network 15 4 Distances Tab The Pipeline Distances tab has only one sub tab The Pipeline Distances Method sub tab is used to choose and configure a method to apply
56. nge the Splits block The Draw Redraw All Splits item redraws the network using all splits including those excluded using the previous menu item The Draw Select Trees item opens the Pipeline Trees Select tab that can be used to select all splits in a given network that are contained in a given set of selected input trees 12 10 Window Menu The Window menu contains the following items The Window About item shows information about the version of SplitsTree4 The Window How to Cite item gives instructions on how to cite the program The Window Nexus Syntax submenu provides the syntax of all Nexus blocks that SplitsTree4 can process The Window Command Syntax item summarizes the syntax of the commands that can be used with the command line mode version of the program or which can be present in an input file The Window Enter a Command item allows entry of a single command 22 e The Window Message Window item opens the message window The bottom of the Window menu contains a list of all open windows 12 11 Configuring Methods By default selecting a menu item that applies some method to the given data such as e g the Trees NeighborJoining will open up a dialog in the Pipeline window which one can use to configure the method by selecting appropriate options For methods that have no options or that are always used with the same options selecting the Don t show this dialog for this method again checkb
57. olean gt Input Characters Output Trees ProteinMLdist This method calculates maximum likelihood protein distance estimates using the following models cpREV 5 Dayhoff JTT mtMAM mtREV24 pmb Rhodopsin and WAG 31 Usage ProteinMLdist Gamma lt double gt Model lt String gt PInvar lt double gt Estimate_variances lt boolean gt Input Characters Output Distances PTreeSplits This method computes the parsimony splits tree 1 Usage PTreeSplits Input Characters Output Splits RecombinationNetwork This method compute a recombination network from binary sequences 22 If the given data is not 0 1 data but DNA then only those sites are considered that show pre cisely two different states The present implementation operates by calling the ReticulateNetwork method Usage RecombinationNetwork MinSplitWeight lt int gt Which lt int gt Method lt String gt 45 ShowSplits lt boolean gt OutGroup lt String gt MaxAngle lt int gt MaxReticulationsPerTangle lt int gt ShowSequences lt boolean gt ShowMutations lt boolean gt Input Characters Output Network ReticulateNetwork This method compute a reticulate network from splits 22 Usage ReticulateNetwork MinSplitWeight lt int gt Which lt int gt Method lt String gt ShowSplits lt boolean gt OutGroup lt String gt MaxAngle lt int gt MaxReticulationsPerTangle lt int gt ShowSequences lt boolean gt ShowMutations lt boolean gt
58. ompute a network using the HybridizationNetwork method The Networks RecombinationNetwork item requests to compute a network using the RecombinationNetwork method Analysis Menu Analysis menu contains the following items The Analysis Bootstrap item opens the Bootstrap dialog The Analysis Show Bootstrap Network item opens a new Main Viewer depicting a network that is based on all splits that occurred in any of the bootstrap replicates Note that this item is enabled only after bootstrapping has been completed The Analysis Show Confidence Network item opens anew Main Viewer containing a network that represents a 95 confidence set for the trees or networks estimated Note that this item is enabled only after bootstrapping has been completed The Analysis Estimate Invariable Sites uses the capture recapture method of 24 The Analysis Compute Phylogenetic Diversity is enabled when a set of taxa are computed It computes the sum of the weights for all splits that separate these taxa into two non empty groups On a tree this is equivalent to the phylogenetic diversity measure of 9 as these splits will be exactly those lying on a path between two taxa The Analysis Conduct Phi test for Recombination tests for recombination using the Phi test of 4 The Analysis Configure Pipeline item opens the Pipeline window which can be used to configure the parameters of a given method The Analysis Configure Recent Methods
59. ones are a b ai a b e c d e c d Figure 1 An unrooted phylogenetic tree A fundamental result in mathematical phylogeny states that any given set of splits corresponds to some phylogenetic tree T with X T 2 if and only if X is compatible 6 Thus any phylogenetic tree may be viewed as a graph whose task it is to give a visual representation of a given set of compatible splits A phylogenetic tree can be thought of as an idealized representation of the historical relationships among a set of taxa and tree building methods are attempts to find a the set of compatible splits that are most consistent with the data according to some algorithm Often there are multiple tree that are equally consistent with the data i e multiple sets of compatible splits In general that collection of splits will be incompatible Moreover there exist inference methods such as split decomposition and Neighbor Net that compute a set of incompatible splits from the data in the form of a given distance matrix Note that split decomposition produces a set of splits that are called weakly compatible while Neighbor Net produces a set of splits that is circular as originally defined in 1 A split network is a more general type of phylogenetic graph that can represent any collection of splits whether incompatible or not 1 For a compatible set of splits it is always possible to represent each split by a single branch and thus the resultin
60. ox will tell the program not to open the dialog in the future but rather to immediately apply the named method Note that configuration dialogs hidden in this way can still be accessed using the Analysis Configure Recent Methods submenu 13 Popup Menus Right clicking on the Main window will open a popup menu There are three different menus that will appear depending upon what is hit by the mouse click If the mouse is clicked on a node of a network then this opens the Node popup menu which has the following items e The Copy Label copies the node label to the system clipboard e The Edit Label opens a dialog to edit the current node label e The Exclude Selected Taxa excludes the selected taxa from all computations e The Show Name labels the selected node by the names of any correspond taxa e The Show Id labels the selected node by the ids of any correspond taxa e The Hide Label hides the label of the selected node e The Configure opensthe Nodes and Edges Nodes tab of the Nodes and Edges window If the mouse is clicked on a edge of a network then this opens the Edge popup menu which has the following items e The Copy Label copies the edge label to the system clipboard e The Edit Label opens a dialog to edit the current edge label e The Show Id labels the selected edge by the id of the corresponding split 23 e The Show Weight labels the selected edge by the weight of the corresponding split e The Show Confidence
61. rom restriction sites using the Nei and Miller method 27 Usage NeiMiller Input Characters Output Distances NJ This method computes the Neighbour Joining tree 28 Usage NJ Input Distances Output Trees 43 NeighborNet This method computes the Neighbor Net splits 5 to produce a Neighbor Net network Usage NeighborNet Variance lt String gt Minimize_AIC lt boolean gt Input Distances Output Splits Noalign This method obtains a trivial sequence alignment by adding gaps to the end of each sequence to make all sequences have the same length Usage Noalign Input Unaligned Output Characters NoGraph This method prevents the program from constructing a final network Usage NoGraph Input Splits Output Network NoSplits This method prevents the program from constructing splits from a set of trees Usage NoSplits Input Trees Output Splits ParsimonySplits This method computes the set parsimony splits 1 Usage ParsimonySplits Input Characters Output Splits PhylipParsimony This method computes the maximum parsimony tree from DNA sequences using an external call to the Phylip package 10 Usage PhylipParsimony PhylipPath lt String gt SearchMode lt String gt NumberOfTreesToSave lt int gt InputOrderSeed lt int gt InputOrderJumbles lt int gt DutgroupRoot lt String gt ThresholdParsimony lt double gt TranversionParsimony lt boolean gt WeightsFile lt String gt Input
62. rtices WIDTH n HEIGHT n SHAPE RECT OVAL FGC color BGC color LINE n VLABELS vertex_id label X xoffset Y yoffset FGC color FONT font vertex_id label X xoffset Y yoffset FGC color FONT font EDGES 1 vertex_id vertex_id ECLASS n FGC color LINE n 2 vertex_id vertex_id ECLASS n FGC color LINE n nedges vertex_id vertex_id FGC color LINE n ELABELS edge_id label X xoffset Y yoffset FGC color FONT font edge_id label X xoffset Y yoffset FGC color FONT font 3 INTERNAL edge_id x y x yl 36 edge_id x y x y 3 END 19 9 Bootstrap Block The Bootstrap block contains the results of a bootstrap analysis It has the following syntax BEGIN ST_BOOTSTRAP DIMENSIONS NTAX number of taxa NCHAR number of characters NSPLITS number of splits FORMAT LABELS LEFT NO SPLITS NO YES ALL YES NO re RUNS the number of runs LENGTH sample length SAME SEED random number seed SAVEWEIGHTS yes no FIXSPLITS yes no COMPUTEDIST yes no OUTPUTFILE file name MATRIX label_1 value_1 split_1 label_2 value_2 split_2 label_nsplits value_nsplits split_nsplits label_nsplits 1 value_ nsplits 1 splits_ nsplits 1 label_n value_n splits_n 3 END 19 10 Sets Block The Sets block can be used to define sets of taxa or characters It has the following syntax BEGIN Sets TAXSET taxset name
63. s dialog is opened using the File Save As item Its purpose is to save the complete state of a document in Nexus format To save parts of the document in Nexus format or in some other supported format listed in Section 20 use the Export dialog 30 18 4 Find Replace Window The Find Replace window is opened using the Edit Find Replace item Its purpose is to find and or replace text Such searches can be performed either in the Main Source tab or in the Main Network tab In the latter case find and replace is performed in all visible node and edge labels Note that changing a node label only changes the label displayed in the network The original taxon label provided in the Taxa block remains unchanged 18 5 Node and Edge Window The Nodes and Edges window is opened using the View Nodes and Edges item Its purpose is to modify the appearance of nodes and their labels and edges and their labels in the displayed network This window has two tabs The Nodes and Edges Nodes tab is used to control the appearance of nodes It can be used to set node size node shape node color and node font Moreover it can be used to determine whether nodes are labeled by taxon names taxon ids or both The Nodes and Edges Edges tab is used to control the appearance of edges It can be used to set edge width edge color and edge font Moreover it can be used to determine whether edges are labeled by split weights split ids split confidences or some
64. simony uninformative 18 ParsimonySplits 20 44 partial trees 12 41 47 Paste 16 Phylip 38 39 44 PhylipParsimony 20 44 Phylogram 22 24 44 PHYML 45 PhyML 20 45 PhyML Path 11 Pipeline 24 pipeline 6 Pipeline Characters 25 Pipeline Characters Filter 25 Pipeline Characters Method 25 Pipeline Characters Select 26 Pipeline Distances 26 Pipeline Distances Method 26 Pipeline Quartets 26 Pipeline Quartets Method 26 Pipeline Splits 26 Pipeline Splits Filter 26 Pipeline Splits Method 26 Pipeline Taxa 25 Pipeline Taxa Filter 25 Pipeline Trees 26 Pipeline Trees Filter 26 Pipeline Trees Method 26 Pipeline Trees Select 26 Pipeline Unaligned 25 Pipeline Unaligned Method 25 pmb 45 PNG 28 Preferences 16 24 28 preferences 28 Preferences Defaults 29 Preferences General 28 Preferences Layout 29 Preferences Status Line 29 Preferences Toolbar 13 29 Previous Tree 11 20 24 Print 15 24 properties file 28 ProteinMLdist 19 45 PTreeSplits 45 Quartets 34 Quit 15 Quit command 48 recombination 9 recombination network 9 12 45 RecombinationNetwork 21 45 Redo 16 Redraw All Splits 22 RefinedBuneman Tree 20 46 Replace 15 Reset 16 17 24 Reset Label Positions 24 Restore All Sites 18 Restore All Splits 18 24 Restore All Taxa 17 24 restriction sites 43 reticulate evolution 9 reticulate network 9 46 reticulate networks 12 ReticulateNetwork 46 Rho
65. submenus lists all recently used methods and can be used to open the Pipeline window in the appropriate tab to configure the parameters of a given method Draw Menu Draw menu contains the following items 21 The Draw EqualAngle item requests to draw a network using the EqualAngle method The Draw RootedEqualAngle item requests to draw a rooted network using the RootedEqualAngle method The Draw ConvexHull item requests to draw a network using the ConvexHull method The Draw Phylogram item requests to draw a tree using the Phylogram method The Draw NoGraph item requests that no network be computed The Draw Select Characters item opens the Pipeline Characters Select tab that can be used to select individual characters that is sites in the given sequence alignment to be displayed on the nodes of the network set of selected input trees The Draw Hide Selected Splits item can be used to remove all selected splits di rectly from the depicted network This does not change the Splits block See also Data Exclude Selected Splits The Draw Hide Non Selected Splits item can be used to remove all non selected splits directly from the depicted network This does not change the Splits block See also Data Exclude Selected Splits The Draw Hide Incompatible Splits item can be used to remove all splits that are incompatible with the set of currently selected splits directly from the depicted network This does not cha
66. t NeighborJoining 23 Trees Next Tree 11 20 24 Trees gt NJ 20 Trees PhylipParsimony 20 Trees PhyML 20 Trees Previous Tree 11 20 24 Trees RefinedBunemanTree 20 Trees TreeSelector 20 Trees gt TreesSelector 11 20 Trees gt UPGMA 20 TreeSelector 20 47 TreesSelector 11 20 trivial splits 8 Type setting conventions 4 naligned 33 ncorrectedP 19 47 ndo 16 31 nix 5 6 pdate command 48 JPGMA 20 47 use Split Selection Mode 29 GC addda Version command 48 View 16 View Auto Layout Node Labels 14 17 View Characters 17 View Collapse All 17 View Distances 17 View Expand All 17 View Flip 17 View Highlight Confidence 17 31 View Invert Selection 14 View Nodes and Edges 5 14 17 31 View gt Reset 16 17 24 View Rotate Left 17 24 View Rotate Right 17 24 View Splits 17 57 View Zoom In 16 24 View Zoom Out 17 24 WAG 45 weakly compatible 9 Weight Threshold 27 Window 22 24 Window About 22 32 Window Command Syntax 22 Window Enter a Command 22 Window How to Cite 22 Window Message Window 23 24 32 Window Nexus Syntax 22 Window Reset 24 Window Reset Label Positions 24 Window Restore All Splits 24 Window Restore All Taxa 24 Window Zoom In 24 Window Zoom Out 24 Windows 5 6 Z closure 41 47 Zoom In 16 24 Zoom Out 17 24 58
67. tates are A then the program will display a Choose Datatype dialog and prompt the user to specify the data type If the current document is non empty then the selected file is opened in a new Main window e The File Open Recent submenu provides access to recently opened or saved files e The File Replace item is used to replaced the current data by a new data set e The File Duplicate item duplicates the current document and opens it in a new Main window e The File Close item closes the current document e The File Save item saves the current document to its file if known e The File Save As item provides a Save As dialog and saves the current document to selected file e The File Export item opens the Export dialog which is used to save individual data in a number of different formats e The File Export Image item opens the Export Image dialog which is used to save the current network in a number of different formats e The File Tools item provides a submenu of tools The File Tools Load Trees item can be used to merge a set of trees into one document This item opens a dialog for opening multiple files The program parses all given files and extracts any trees found in them and opens a new Document containing all trees found The File Tools Load Multi Labeled Tree item can be used to read a single tree containing multiple labels and to convert it into a single labeled split network and will be made avai
68. taxa e Unaligned unaligned sequences e Characters aligned character sequences e Distances pairwise distances between taxa e Quartets possibly weighted quartet topologies e Trees list of possibly partial trees e Splits possibly weighted splits e Network phylogenetic tree or network e ST_Assumptions contains all methods and options used to compute data e ST_Bootstrap bootstrap support of splits The first eight blocks Taxa Unaligned Characters Distances Quartets Trees Splits and Network are organized as a pipeline and data is processed from left to right along this pipeline Any non empty document must contain a Taxa block and will usually contain an ST_Assumptions block All other blocks are optional and the presence or absence of some block depends on the set of computations that the user has selected We will use the term source block to denote the left most block in the pipeline excluding the Taxa block The source block contains the original data that is provided to the program Any computations performed by the program will update blocks from left to right along the pipeline starting after the source block Note that some types of computations do not fit into this pipeline design for example Splits Tree4 cannot provide any method that takes a Splits block and produces a Trees block because the latter occurs before the former in the pipeline Typically the user will provide a source block and will the
69. that the program supports see Section 20 e once the data has been executed the source data is displayed in Nexus format and e if an error is encountered while parsing an input file the file is opened and the line in which the error was detected is selected 10 4 Tool bar The tool bar associated with the window provides buttons for quick access to many of the menu items It can be configured using the The Preferences Toolbar tab 11 Graphical Interaction with the Network We now describe how the user can interactively modify the layout and attributes of the displayed network The View menu is used to rotate move and zoom in and out Alternatively the view can be changed using a wheel mouse together with the following modifier keys 13 none zoom in and out ALT option rotate Command apple move horizontally Command apple amp ALT option move vertically Here are additional modifications that can be performed e The graph can be dragged around by clicking and dragging a node e If all selected nodes lie on one side of a band of parallel branches representing a single split then clicking and dragging on one of the nodes will change the angle of the branches e By default clicking on an edge will select all edges representing the same split and all nodes on the smaller side of the represented split To select all nodes on the other side of the split use the View Invert Selection menu item To allow selection of
70. the presence of hybridization events such as allopolyploidization or diploid hybridization and in both cases the net effect is that a new species arises that has obtained some of its genes via one ancestral species and the other genes via another ancestral species A recombination network is used to describe evolution in the presence of recombination events As presently implemented in SplitsTree4 such recombination networks can only be computed for binary sequences Such sequences may for example indicate the presence or absence of a certain restriction site at a certain position There are two main differences between hybridization networks and recombination networks First the former are based on gene trees whereas the latter are based on sequences Second a recombination network is additionally annotated by inferred sequences at each of the nodes and the mutations that happen along each of the branches Moreover the recombination nodes are usually annotated with a description of which parts of the two ancestor sequences are used in the recombination event In contrast to split networks these types of networks can be directly interpreted in evolutionary terms Recent research has revealed that there are strong connections between split networks and reticulate networks 21 22 and these are used in SplitsTree4 to provide methods for computing reticulate networks A more detailed discussion of the fundamental concepts can be found in 17 and
71. tstrap Network 21 Analysis gt Show Confidence Network 21 Assume command 48 Auto Layout Node Labels 14 17 batik 49 binary 9 Binary2Splits 39 BIONJ 20 BioNJ 39 blocks 6 BMP 28 Bootstrap 21 31 37 Bootstrap command 48 box opening 41 branch 8 BunemanQuartets 39 BunemanTree 20 39 Characters 17 33 Choose Datatype 15 30 circular 9 Clear All Taxa Sets 16 Close 15 Closest Tree 27 ClustalW 38 39 Coalescent 40 coalescent method 40 Collapse All 17 Command Syntax 22 command line mode 48 command line options 47 compatible 8 Compute Phylogenetic Diversity 21 Concatenate Sequences 15 concatenate sequences 15 Conduct Phi test for Recombination 21 Confidence Threshold 27 Configure 23 24 Configure Pipeline 5 11 21 24 Configure Recent Methods 5 11 21 23 consensus network 12 40 consensus trees 40 ConsensusNetwork 20 40 ConsensusTree 11 20 40 constant 18 Convert text to graphics 28 Convert to Nexus 18 ConvexHull 22 40 Copy 16 Copy Label 23 cpREV45 45 create a node 29 create an edge 29 Cut 16 Data 17 32 data type 15 Data Convert to Nexus 18 Data Exclude Constant Sites 18 Data Exclude Gap Sites 17 Data Exclude Parsimony Uninformative Sites 18 Data Exclude Selected Splits 18 22 52 Data Exclude Selected Taxa 17 Draw Hide Incompatible Splits 22 Data Execute 18 Draw Hide Non Selected Splits
72. u The Edit gt Cut item is used to cut text The Edit Copy item is used to copy text or to copy the current network as an image The Edit Paste item is used to paste text The Edit Select All item is used to select all nodes and edges of a network The Edit Invert Selection item is used to invert the selection of nodes The Edit gt Taxon Sets submenu is used to select all nodes labeled by a given taxon set or to add new taxa sets to the SETS block The SETS block can be copied to other files to provide a convenient method to quickly label or select taxa in different taxa groups The Edit gt TaxonSets gt A11 item selects all taxa The Edit gt TaxonSets New taxa set item creates a new taxa set with the currently selected taxa and a name provided by the user Note that the name should be a valid NEXUS name If a set exists with this name it is overwritten The Edit TaxonSets Clear All Taxa Sets removes all taxa sets from the SETS block The Edit Find Replace item opens the Find Replace dialog The Edit gt Go to Line item is used to scroll to a particular line in the Main Source tab The Edit Preferences opens the Preferences window 123 View Menu The View menu contains items that control aspects of the visualization of a network This menu changes depending on which tab on is in In the Main Network tab the View menu contains the following items which are all undo able and redo able e The V
73. urce text area displayed in the tab e The Data gt Execute item parses the text in Nexus format and executes it e The Data Convert to Nexus item parses the text in any of the supported file formats see Section 20 including old Nexus and converts it into Nexus format and replaces the text in the source text area by the converted text If either of the first two items is used and the text contained in the text area is not in Newick format then a dialog is displayed that allows the user to request to import the text before parsing it 18 The Data gt Load item is particularly useful if you would like to load a large data set and would like to prevent the program from performing any initial calculations First open the file ausing the File Open item while in the Main Source tab then use the Data Load item to load the data into the program and then switch to the Main Network No calculations will be performed until you choose some menu item 12 5 The 12 6 The Distances Menu Distances menu contains the following items The Distances UncorrectedP item requests the program to compute distances using the UncorrectedP method The Distances LogDet item requests the program to compute distances using the LogDet method The Distances HKY85 item requests the program to compute distances using the HKY85 method The Distances gt JukesCantor item requests the program to compute distances using the
74. us the program will often be able to parse it as it contains code to automatically convert from old to new Nexus format However the algorithm that does this does not provide a full implementation of the old Nexus format and thus sometimes it will be necessary to reformat a file by hand It is easy to tell the difference between old Nexus and new Nexus if a file in Nexus format contains a Taxa block then it is new Nexus Most blocks in both formats have the same name and similar syntax One main difference is that the Characters block in new Nexus is called a Data block in old Nexus In the following syntax descriptions we used upper case letters for keywords square brackets for optional statements and curly brackets to indicate a list of choices 19 1 Taxa Block The Taxa block is the only mandatory block in a Nexus file Its purpose is to list the names of all taxa It has the following syntax BEGIN TAXA DIMENSIONS NTAX number of taxa TAXLABELS taxon_1 taxon_2 taxon_ntax TAXINFO info_1 info_2 info_ntax END 32 The TAXLABELS statement is optional if it is followed by a source block that contains all taxa labels 19 2 Unaligned Block The Unaligned block contains unaligned sequences It has the following syntax BEGIN UNALIGNED DIMENSIONS NTAX number of taxa FORMAT DATATYPE STANDARD DNA RNA NUCLEOTIDE PROTEIN RESPECTCASE MISSING symbo1 SYMBOLS symbol symbol LABELS LEF

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