Manual - Copycat
Contents
1. Auch Daniel H Huson Markus G ker Copycat Co phylogenetic Analysis Tool Bioin formatics 23 7 898 900 2007 1Of course any kind of associations with hosts can be examined in that way including mutualists We refer to parasites only just for convenience 1 Checklist In prior to installing Copycat please make sure that you have verified the following items You have at least one of the following operating systems Mac OS X tested on 10 6 8 a Win32 compatible or a Linux based operating system currently tested under Windows XP Windows 7 and Linux x86 32bit with GTK 2 0 Your machine is equipped with at least 512 MB of memory Even though it is not recommended hardware with less memory can sometimes be used as long as the NCBI taxonomy facilities are not applied The Java 1 5 or higher runtime environment is installed and the Java Binary must be included in the PATH environment variable this is done automatically by the Java Installer for Windows Java 1 5 a k a JDK5 is available here http java sun com javase downloads index jsp Hint by entering the command java version in a command console the cur rently installed version is reported The Mac version additionally requires the GNU scientific library GSL installed A binary installer can be found on http ascend4 org Binary_installer_ for_GSL 1 13_on_Mac_O5_X Optional You have downloaded the curre2. Figure 6 The second tab of Copycat DistPCoA supports two correction methods for negative eigenvalues Lingoes method and Cailliez method The user might choose one of these or simply select the no cor rection method option The advantages and disadvantages of the several corrections methods are explained in e g 3 and 6 pp 432ff 4 2 Step 2 The user selects an association file 4 3 Step 3 create host distance matrix The user has three possibilities e The user selects a pre existing host distance matrix e The user specifies a list of host taxa which is used to reconstruct the NCBI host tree The tree is automatically converted to a patristic distance matrix 2 11 e The user specifies a host tree which is used to create the distance matrix Once a tree is constructed several of its features are reported to the message win dow Balance is a measure of tree balance as described by I Note that balance can only be computed for rooted binary trees Cherry is the measure of tree balance suggested by 7 divided by the maximum possible number of cherries n 2 provided that n is the number of taxa Resolution described as Colless consensus fork index in 8 as well as information content 9 are measures of topological reso lution Resolution is just the number of internal nodes divided by the maximum possible number of internal nodes i e n 2 and thus bound
3. between 0 and 1 A value of 1 indicates full resolution The cladistic information content has some theoretical advantages but in case of not fully resolved topologies this measure may rapidly con verge towards 0 if the number of taxa increases and thus may not be applicable when dealing with large datasets N B Due to the presumed trade off between the number of taxa and the topological resolution available as input for Parafit the user has to base her decision whether to conduct a Parafit analysis based on taxonomic data not only on the number of resolved associations but also on the amount of topological resolution Even though one of the advantages of Parafit is that it does not require fully resolved binary trees 5 trees may well display not enough topological resolution too many polytomies to be of value in conducting cophylogenetic analyses As an extreme example consider a totally unresolved taxonomic tree Since in that case the eigenvectors of all taxa as output by DistPCoA will be identical such a tree will lead to all associations being insignificant just for trivial reasons 4 4 Step 4 create the parasite distance matrix This is the same as above 4 5 Step 5 validation If all input files have been specified and all parameters been set the user should then hit the validate the specified data button During this validation the program reports whether the taxon names in the association matrix are consistent
4. data Users Jan Dropbox Copycat_Examples Ere Path to the taxonomy files pse Workspace Java Workspace Copycat2_Swing CopycatFiles T Reset C Apply Ok Cancel e aa Figure 2 Copycat s configuration dialog the configuration is not complete the dialogue will be displayed again until everything is well configured Now the Copycat application launches It generates a random session ID here ID 9511 and creates a subdirectory for this session within your working directory e g D Copycat myworkingdir ID 9511 All files and results are now going to be stored in that directory The contents of that folder and thus the results of this session can be examined by selecting the View menu item 3 Copycat The first tab The first tab see Fig 3 deals with the creation and pre processing of an association table containing parasite or host associations 3 1 Finding NCBI taxonomy IDs Given an unresolved association list containing parasite or host names the Resolve Association File option tries to assign a NCBI taxonomy ID for each organism name The set of IDs is necessary for inferring the NCBI host or parasite tree The user specifies a file unresolved association table containing one parasite and one host name per line tab separated Then Copycat tries to retrieve an NCBI taxonomy ID equivalent to each entry e g the taxonomy ID 9606 for the host name Homo sapiens These resul
5. process has finished by 1 displaying Resolving step finished successfully the organism name followed by the taxonomy ID e g Homo sapiens 9606 The user should ascertain that this taxonomy ID is definitely contained in the NCBI taxonomy database The better and most consistent way of retrieving a correct ID is as follows the user corrects the organism name and then applies the changes by pressing the apply button By entering a name available in the NCBI taxonomy the respective organism field in the association table should then yield the corresponding ID Finally all resolvable associations are written to a file after having selected the dump results to working directory button option Along the new association file two files containing only the parasite and host names are written to the same directory 3 1 1 Characteristics of the association table The user hits the select association file button and then specifies an association table AT refer to first step The AT is read and the following features are reported to the message window e The distribution of the taxonomical ranks within the set of hosts and the set of parasites respectively in accordance to the NCBI taxonomy e The affiliation of each parasite and host taxon respectively to one of the follow ing divisions division 0 Archaea division 1 Bacteria division 2 Eukaryota division 3 Viroids division 4 Viruses divisi
6. 7 P Legendre and M J Anderson Distance based redundancy analysis testing multi species responses in multi factorial ecological experiments Ecological Mono graphs 69 1 24 1998 P Legendre and M J Anderson Program distpcoa 1998 Pierre Legendre Yves Desdevises and Eric Bazin A statistical test for host parasite coevolution Syst Biol 51 2 217 234 April 2002 P Lengendre Numerical Ecology 2nd English edition Elsevier Amsterdam Amsterdam 2nd englis edition 1998 A McKenzie and M Steel Distributions of cherries for two models of trees Mathematical biosciences 164 1 81 92 2000 DL Swofford When are phylogeny estimates from molecular and morphological data incongruent Phylogenetic analysis of DNA sequences Oxford Univ Press New York pages 295 331 1991 J L Thorley and R D M Page RadCon phylogenetic tree comparison and con sensus Bioinformatics 16 5 486 2000 K Vanky European smut fungi G Fischer Stuttgart Jena New York 1994 K Vanky European smut fungi Ustilaginomycetes pp and Microbotryales ac cording to recent nomenclature Mycologia Balcanica 2 169 177 2005 20
7. 75 9 0 01134 0 01248 no 7 Save As Cancel Figure 5 Copycat s representation of the Broken Stick Distribution 3 2 Computation of the broken stick distribution BSD for the set of resolved associations The user selects a resolved AT which results in a new window displaying the BSD see Fig B It basically consists of two parts the first part shows the BSD for the parasites indicated by a P in the first column of each line the second part the BSD for the hosts a H as identifier The further columns show the rank of the taxon its name its absolute and relative frequency of occurrence within the associations and its expected frequency according to the BSD The last column shows whether the real relative frequency is larger than the expected one By holding the CTRL button Mac OS X Command button and using the left mouse button the user can highlight multiple entries within the list Each entry represents a parasite or host which is then marked for removal from the association table Often the entries in the list are highlighted by means of an alternating pattern of dark and light grey Several lines sharing the same colour scheme correspond to the same tied rank and therefore have to be treated equally N B The broken stick distribution 6 p 244 is a standard null model of community structure in ecology It can be used to predict species relative abundances but may also be used with other kind of da
8. Copycat The Cophylogenetic Analysis Tool User Manual March 12 2012 Contents 1 Checklist 4 4 Copycat The first tab 5 3 1 Finding NCBI taxonomy IDs 000 5 3 1 1 Characteristics of the association table 8 3 2 Computation of the broken stick distribution BSD for the set of re 3 3 Filtering of an association table 2 0 0 0 0008 10 4 Copycat The second tab 10 A1 pela a waved eek ee a Be eed oO ORE A SLES Gee 10 Ce pee a Saw haw ee Be Ree oe hah ee we ee ed 11 4 3 Step 3 create host distance matrix 0004 11 4 4 Step 4 create the parasite distance matrix 12 4 5 Step 5 validation a 12 5 Copycat The third tab 13 6 Copycat Available menu bar options 13 13 13 13 16 6 2 1 View content of working directory 16 6 3 Options cee ete ve tev eevee eee eee eey 16 6 3 1 Enable Strict Filtering of Association Table 16 6 3 2 Use Equal Branch Length 1 for tree2dist Conversion 16 Use AxPcoords and AxParafit instead of DistPCoA and Parafit Seb ped ab Bete dea ede Ga A Pk deed Gee PA Lee ae dh a 17 6 4 1 Show setup menu at next program start 17 7 Tutorial A step by step example run of Copycat 17 host A parasite H host B q lt parasite G host C bid parasite F link significant host D parasite E F
9. Environmental samples Associations Level of Detail optional Leave as is O Compress Associations to the Following Rank Removal of Specific Taxa optional Enter host parasite taxa which shall be removed Generation of Both Host and Parasite Taxa Lists Welcome to CopyCat The cophylogenetic analysis tool INFO The operating system is MAC OS X INFO The working directory is Applications Copycat DefaultWorkingDir Figure 3 The first tab of Copycat AAA Copycat File View Options Setup Help 1 Preprocessing of Input Data 2 Configuration and Execuation of AxParafit 3 Evaluation of AxParafit Results Step 1 Resolved Associatio E Results 794 associations have been resolved Step 3 Exclus Ol OW Assod O Remol Entei Gener 4 SaveAs Cancel f Welcome to CopyCat The cophylogenetic analysis tool INFO The operating system is MAC OS X INFO The working directory is Applications Copycat DefaultWorkingDir 794 associations resolved Resolving step finished successfully Figure 4 An association table as resolved by Copycat The green entries have a valid ID assigned for both parasite and host as provided by the NCBI Taxonomy Database Unresolved associations are marked red The message window at the bottom informs the user once the resolving
10. at session ID The time the wrapper is running any interaction with Copycat is blocked De pending on the size of the input data the Parafit run can be a timeconsuming issue Analysis of the Parafit results Parafit performs tests for both the overall phylogenetic congruence as well as the significance of individual associations These results are listed in the file HostPara out Copycat reports the location of HostPara out by printing WRAPPER Please check file D Copycat defaultwdir ID_3738 ID_8313 Hostpara out for results HostPara out holds the results on the individual links in the following format Parasite 17 Host154 F1 Probl 0 94600 F2 0 00016 Prob2 0 99200 As Parasite 17 and Host 154 provide no information on the actual organisms Copycat needs to know the location of the parasite and the host distance file used in that specific Parafit run These files should reside in your working directory Once the three files Hostpara out parasite distance matrix and host distance matrix have been specified Copycat opens a new window showing the results An example is shown in Figure 8 19 References 1 D H Colless Review of Phylogenetics The Theory and Practice of Phylogenetic Systematics by E O Wiley Systematic Zoology 31 1 100 104 1982 J S Farris The meaning of relationship and taxonomic procedure Systematic Zoology pages 44 51 196
11. ata This tab is divided into 4 steps 4 1 Step 1 The number of permutations per row of the association matrix to be conducted by Parafit can be specified Parafit requires principal coordinates inferred from host and parasite distance matrices as computed by e g the program DistPCoA 4 10 File View Options Setup Help a lt P Preprocessing of Input Data 2 Gonfiguratiomand Execuationof AxParafit 3 Evaluation of AxParafit Results n Step 1 Choose Your AxParafit AxPcoords Executable AxPcoords Executable AxPcoords_mac_gsl exe r AxParafit Executable AxParafit_mac_ATLAS exe 4 Step 2 Specify Parameters for AxParafit AxPcoords Number of Permutations Used in AxParafit 99 i Correction Method Used in AxPcoords 3 no correction Step 3 Specification of Association File Select Association File Step 4 Generation of Host Distance Matrix Create Distance Matrix From Host Taxa List C Select Input File Step 5 Generation of Parasite Distance Matrix Create Distance Matrix From Parasite Taxa Select Input File Step 6 Input Data Validation Step 7 Analysis Validate Start Local Analysis OR Pack All Required Files for Remote Analysis Welcome to CopyCat The cophylogenetic analysis tool INFO The operating system is MAC OS X INFO The working directory is Applications Copycat DefaultWorkingDir
12. atrix is finished once the following three lines appear at the end of the message window here example values INFO on parasite tree Resolution 0 252525 INFO on parasite tree Balance input is no rooted binary tree INFO on parasite tree Information content Infinity The working directory should now contain the files parasites dist and para sites out tree These info messages show the resolution and the phylogenetic information content which cannot be computed for large numbers of taxa of the host parasite tree created If the resolution is not satisfying enough you have the option to cancel the further cophylogenetic analysis at this stage If you want to proceed you have to choose the validate button The validation step ensures that the taxa contained in the following three files are consistent with respect to each other This means that a parasite s name contained in the association table should exist in the parasite distance matrix and so forth If the validation is successful you will have the option to immediately start the Parafit run on this machine or as an alternative to pack all relevant files to a zip archive This archive can be transferred to another probably more powerful machine Once the ParafitWrapper has started either on this or another machine it generates a random session ID similar to Copycat and creates a respective subdirectory within your working directory This directory is named after th
13. ds for negative eigenvalues are not supported by AxPcoords 6 4 Setup 6 4 1 Show setup menu at next program start If you want to change the working directory you just have to check this option On the next start of Copycat the configuration dialogue will appear again 7 Tutorial A step by step example run of Copycat This tutorial focuses on the kind of input needed for certain steps in Copycat and shows the output produced by that input The underlying data set for this example run is the list of European smut fungi and their hosts from Vanky M This data set does not contain parasite or host trees or distance matrices so we have to construct them using the NCBI taxonomy 1 The input An association table of smut fungi parasites and their respective hosts Here is an excerpt of the input file smut_fungi_association_table txt 2 Resolving the association table Each parasite host contained in the NCBI Tax onomy Database has a unique taxonomy ID This step tries to gather these IDs Each association containing both a parasite and a host with a valid ID is used in the resulting so called resolved association table A representation of an unresolved association table is shown below lht 3 Generation of the parasite host taxa lists For the Parafit analysis 5 of this association data we first need to draw two lists from the resolved asso ciation table one containing all parasite taxa and another one conta
14. e 7 deals with the evaluation of the Parafit results After the analysis has ended an output file called Parafit out should have been created In this step this output file is specified via the open dialogue together with the host and parasite distance matrix used in that Parafit run The distance matrix files are needed to display the correct organism names instead of the non interpretable labels like Parasite 4 or Host 17 A sample Parafit output as resolved by Copycat is shown in Figure 8 6 Copycat Available menu bar options The menu bar offers the following options 6 1 File 6 1 1 Transfer content of working directory to a place of your choice Once your work with Copycat is done you might want to transfer data from the working directory to a directory of your choice This can be done by selecting this option 6 1 2 Download NCBI taxonomy file s As mentioned above it is advisable to get the latest NCBI taxonomy file from time to time The taxonomy itself is steadily improved due to the incorporation of more recent phylogenetic insights and the total number of both terminal taxa and taxa of higher rank included in the taxonomy dump files is steadily increasing potentially increasing both the number of resolved associations and the topological resolution in the NCBI based cophylogenetic analyses By checking this option Copycat downloads the latest NCBI taxonomy dump file and places i
15. ining all host taxa This is achieved by selecting apply settings to association list Here the specified association list can be filtered in regard to certain criteria 3AMD Core Math Library 4Intel Math Kernel Library 17 parasites hosts Anthracoidea altera Anthracoidea angulata Anthracoidea arenaria Anthracoidea arenaria Anthracoidea arenaria Anthracoidea arenaria Anthracoidea arenaria Anthracoidea aspera Anthracoidea aspera Anthracoidea aspera Anthracoidea aspera Anthracoidea baldensis Carex fuliginosa Carex hirta Carex arenaria Carex brizoides Carex ligerica Carex ovalis Carex praecox Carex appropinquata Carex chordorrhiza Carex diandra Carex glareosa Carex baldensis Table 1 The unresolved association table Parasites are in the left column hosts in the right column Each parasite is separated from its respective host by a tab character The file contains 1853 host parasite associations and as a side effect the parasite host taxa lists are written to the working di rectory Naturally the user is not obliged to select certain filter criteria but can simply choose the leave associations in their current state option Con sequently the specified association table stays untouched In this tutorial we make use of the latter and issue the association table gained in the previous section This operation will take a moment Finally the following two files appear in the wor
16. king directory hosts_filtered_using_option_0 txt and para sites_filtered_using_option_0 txt The value 0 indicates that we selected the leave associations in their current state option 4 Creation of a host distance matrix and a parasite distance matrix The taxa lists from the previous step are now being used for the creation of the re spective NCBI host tree and NCBI parasite tree Once this is done the re spective distance matrices are generated We switch to Copycat s second tab and select the distance matrix from host taxa list option together with the hosts_filtered_using_option_0 txt file This results in the call of the Paraf it Wrapper The wrapper will now try to create the denoted host distance matrix You might want to follow the process of the wrapper by reading the lines in the message window that are marked by the purple WRAPPER tag The wrapper is fin ished once the message window contains the following three information lines at the very end here example values INFO on host tree Resolution 0 252525 INFO on host tree Balance input is no rooted binary tree INFO on host tree Information content Infinity The working directory should now contain the files hosts dist and host out tree 18 We repeat the procedure for the parasites_filtered_using_option_0 txt file by se lecting the distance matrix from host taxa list option in the box below The distance m
17. link not significant host E parasite D Figure 1 A tanglegram showing host parasite associations between both five parasite and five host taxa Copycat is a software tool written in Java which provides an easy and fast access to cophylogenetic analyses It incorporates a wrapper for the pro gram Parafit which conducts statistical tests for the presence of global congruence between host and parasite phylogenies and for the significance of individual host parasite associations A tanglegram is the visualiza tion of host parasite associations and can be further enhanced by adding Copycat s information on non significant links see Fig i The software offers various features such as the creation of customized host parasite association data the reconstruction of host or parasite trees from the NCBI taxonomy and the computation of several tree statis tics As of April 2007 Copycat supports Alexandros Stamatakis programs AxParafit and AxPcoords which are highly optimized versions of Parafit and DistPCoA respectively see section Copycat Available menu bar op tions This manual describes the features specific to Copycat regarding the principles of the statistical tests implemented in Parafit users are strongly advised to consult 5 The literature cited in the references sec tion is also suggested for further reading If you use Copycat you should cite the accompanying paper Jan P Meier Kolthoff Alexander F
18. mic group but based on character data as well as the resolution of the host or parasite taxonomy trees as described below LeO Resolved Association File Results type rank taxon frequency proportion expected prp gt exp P 1 Ustilago_striiformis_307781 60 0 07557 0 03499 yes r P 2 Microbotryum_violaceum_5272 42 0 0529 0 02881 yes oO P 3 Schizonella_melanogramma_63386 39 0 04912 0 02573 yes P 4 Jamesdicksonia_dactylidis_63287 31 0 03904 0 02367 yes P 5 Thecaphora_saponariae_72562 25 0 03149 0 02213 yes P 6 Urocystis_ranunculi_63394 23 0 02897 0 02089 yes P 7 Ustilago_bullata_117172 21 0 02645 0 01986 yes P 8 Tranzscheliella_hypodytes_349358 20 0 02519 0 01898 yes P 9 Entyloma_ranunculi repentis_189607 20 0 02519 0 01821 yes P 10 Entyloma_hieracii_189602 16 0 02015 0 01752 yes P 11 Entyloma_microsporum_62642 15 0 01889 0 01691 yes P 12 Anthracoidea_karii_265844 14 0 01763 0 01635 yes P 13 Stegocintractia_luzulae_86812 ll 0 01385 0 01583 no P 14 Ustilentyloma_brefeldii_355612 11 0 01385 0 01536 no P 15 Microbotryum_stellariae_288790 ll 0 01385 0 01492 no P 16 Ustilago_avenae_120650 11 0 01385 0 0145 no P 17 Anthracoidea_subinclusa_265861 10 0 01259 0 01412 no P 18 Anthracoidea_fischeri_251615 10 0 01259 0 01376 no P 19 Microbotryum_dianthorum_72560 10 0 01259 0 01341 no P 20 Thecaphora_thlaspeos_469304 10 0 01259 0 01309 no P 21 Vankya_ornithogali_437798 10 0 01259 0 01278 no a P 22 Entorrhiza_casparyana_633
19. nt NCBI taxonomy file taxdmp zip from the following URL Ftp ftp ncbi nih gov pub taxonony taxdmp 2p and placed this file in the input data subfolder It is located in your Copycat installation folder e g home john Copycat input data As the NCBI taxonomy is updated on a regular basis it is advisable to get the lat est version of taxdmp zip from time to time The Copycat Download already ships with an older version of the NCBI taxonomy data 2 The first start Once you have started Copycat for the first time a configuration dialogue will appeall if this dialog won t show up you can access it by choosing the Setup option from the top menu see Fig p Here you might want to choose another working directory WORKING_DIR or another directory for your custom data USERDATA If you are experiencing problems while alternative settings you might want to default to the well tested stan dard settings The latter will be relevant if Copycat asks for a file to open then this directory will be prompted first To proceed please click apply followed by save and proceed By pressing exit Copycat the whole application will close If 2The following screen shots were made using the Mac version of Copycat the look amp feel of the Windows and Linux versions differs slightly vere Setup Menu Your Copycat working directory T Path to your input
20. on 5 Other division 6 Unclassified division 7 taxon ID not found in NCBI taxonomy e The number of associations contained in the AT e The number of different parasite taxa in AT e The number of different host taxa in AT e The estimated size of the association matrix drawn from the AT N B As a rule a phylogenetic tree derived from character data e g molecular sequences contains more information than a taxonomy of the same taxa since the topology of the latter is usually much less resolved However if a cophylogenetic study is based on specific marker sequences such as 16SrRNA or ITS it is limited to species for which there is a common marker gene available Even though the number of single locus or even genome sequences is steadily increasing we presume that NCBI taxonomic data are available for many more taxa than are such orthologous genetic data We guess this rule also holds if we consider non homologous loci for use in supertree reconstruction let alone the current debate about whether and how to infer supertrees However this may not be true for all taxonomic groups Since there most likely is a trade off between the number of taxa and the topological resolution available as input for Parafit the user has to decide whether a certain Parafit analysis based on taxonomic data is worth conducting or not It is therefore necessary to closely examine the number of resolved associations compared to those of a study of the same taxono
21. oords and AxParafit instead of DistPCoA and Parafit By selecting this option AxPcoords and AxParafit are used instead of Legendre s programs 5 Copycat supports Alexandros Stamatakis programs AxParafit and AxPcoords which are highly optimized versions of Parafit and DistPCoA respec tively Copycat searches within the code subdirectory which contains all executa bles for the analyses By default this folder contains the following operating system dependant executables e Parafit_win exe e DistPCoA_win exe e AxParafit_win exe e AxPcoords_win exe 16 The attribute win denotes the executables for the Windows platform accordingly mac for Macintosh and linux for Linux systems are used In case the user has se lected the above option Copycat chooses AxParafit_win exe and AxPcoords_win exe operating system Windows If you wish to use externally compiled executables e g using the ACMI or MKI libraries they should obey to the naming convention PROGRAM _ OS _ LIBRARY e g AxParafit_win_ MKL exe or AxPcoords_win_ MKL exe and moved into the code subdirectory Copycat will detect and use them for the next computation Such an optimized executable will always be preferred to the de fault one Once the user has selected the AxPcoords AxParafit option the option correction method used in DistPCoA the second item on the second tab will be disabled because correction metho
22. ovides an objective means to distinguish these from normal species 3 3 Filtering of an association table This step allows the selection of divisions as defined within the NCBI taxonomy whose corresponding taxa and hence associations should be completely removed from the table The additional filter option provides a so called rank mapping feature Each taxon is mapped to its respective parent taxon until the specified rank e g genus or family is obtained Redundant entries are removed automatically The field remove parasite host associations containing specific parasite host taxon IDs allows the removal of associations whose taxa are listed in this box Valid input is a list of space separated taxa IDs each of them with the leading letter P or H indicating the taxon s membership in the group of parasite or host taxa the taxa being selected via the BSD option are listed in this field For example H9606 would result in a removal of those associations having a host associated with the taxonomy ID 9606 Homo sapiens_9606 would also be a valid input although only the ID is of interest 4 Copycat The second tab This tab see Fig 6 deals with the selection of the input files and parameters for Parafit 5 and with the Parafit run itself Here the most important feature is a wrapper for the program Parafit preparing and finally providing the properly for matted input d
23. t Filtering of Association Table By default the filter process scans an association table and removes all associations lines which do not fulfill one of the following criteria Both parasite and host label have to exist in the NCBI taxonomy and should not be blacklisted If the user has provided an association table containing custom taxon labels such as Patient234 instead of Homo sapiens the program would remove this line from the new associ ations table due to the first condition have to exist in the NCBI taxonomy Even though this condition can be relaxed by unchecking the option Enable Strict Filtering of Association Table in the menu bar 6 3 2 Use Equal Branch Length 1 for tree2dist Conversion If you have specified a tree file in Copycat s second tab you might want to have topological distances in the distance matrix resulting from that tree By checking this option branch lengths are set to 1 By default this option is not checked In that case a patristic distance matrix results in which each pairwise distance between two taxa A and B represents the sum of the number of taxa in which A is contained including A but not B and the number of taxa in which B is contained including B but not A Accordingly by default branch lengths in the NCBI taxonomy tree may be larger than one representing more information extracted from the taxonomy than just the topology 6 3 3 Use AxPc
24. t in the appropriate directory 13 File View Options Setup Help 1 Preprocessing of Input Data 2 Configuration and Execuation of AxParafit Step 1 Select the Parafit Distance File Used in the Parafit Run Step 2 Select the Host Distance File Used in the Parafit Run Step 3 Select the File Containing the Parafit Results i e HostPara out rStep 4 View on the Final Results Welcome to CopyCat The cophylogenetic analysis tool INFO The operating system is MAC OS X INFO The working directory is Applications Copycat DefaultWorkingDir Figure 7 The third tab of Copycat 14 ane Evaluation of AxParafit Results AxParafit Results Overall cophylogenetic structure is highly significant 0 0101 lt 0 02 sig val 599 lir Value of Significance 0 02 assoc no parasite host probl value prob2 value Save As Cancel Figure 8 Copycat s representation of the Parafit output By selecting dump in formation to working directory the results are stored in a simple text file ASCII format Lines contained in that file marked by a character 9 represent significant links while lines starting with a sidered as non significant 15 character are con 6 2 View 6 2 1 View content of working directory This shows a view of the current working directory and its content 6 3 Options 6 3 1 Enable Stric
25. ta such as e g eigenvectors 6 p 410 Species the relative frequency of which is larger than the corresponding broken stick value occur more frequently than expected by chance We have included the BSD here since it may be used to detect host or parasite species which are represented in significantly more associations than others This is not to say that Parafit is unable to deal with widespread parasites on the contrary these are treated more consistently in Parafit than in other cophylogeny programs 5 However a list of associations derived from literature data may for instance include many more associations from host species which are medically or economically impor tant and thus have been studied more intensively than their less important relatives If the BSD detects species which are represented in a particularly large number of associations the user may wish to conduct Parafit runs both before and after ex clusion of these taxa In case such taxa display a cophylogenetic behaviour strongly deviating from that of other taxa i e significant vs insignificant associations or vice versa presence or absence of these highly frequent taxa may considerably influence global significance Even though Parafit s results will according to Legendre et al 2002 always be the correct ones given the correctness of the associations the user may be interested in the impact of such extremely widespread parasites The brokenstick method pr
26. ts will be automatically displayed in an extra window see Figure 4 If that window has accidentally been closed it will immediately show up again once the user has specified the unresolved association file table again und Select Association File If for both parasite and host name a respective taxon ID is retrieved this parasite host association appears in the final resolved association table as a green colored entry The set of all host or parasite taxa contained in that table is used for the reconstruction of the NCBI host or parasite tree respectively If a certain parasite or host name can t be resolved e g because of a misspelling the user has the possibility to manually enter a proper NCBI taxonomy ID The format for this is File View Options Setup Help H Preprocessing of Input Data 2 Configuration and Execuation of AxParafit 3 Evaluation of AxParafit Results l Step 1 Resolve Association File optional Select Association File Step 2 Statistics optional Compute Distribution of Host Parasite Taxa Select Association File Show Broken Stick Distribution Select Association File Step 3 Filter Parasite Host Associations Creating Parasite Host Taxa Lists Exclusion of Certain Systematic Divisions by NCBI Definition optional Bacteria Invertebrates Mammals Phages C Vertebrates Viruses Plants O Primates C Rodents M synthetic Unassigned
27. with the taxon names in the host and parasite distance matrix or not If taxon names are present in the association table which are not contained in the respective distance matrix the program returns an error In the opposite case taxon names from the distance matrix can t be found in the association table the program offers a shrink distance matrix option which allows the removal of the respective columns and rows from the distance matrix N B In case host or parasite trees are derived by pruning from larger phylogenies it is much more convenient to change just the association table than to manipulate the trees themselves This feature of Copycat results in a great gain in user flexibility with respect to running Parafit with slightly different sets of taxa If the validation returns no errors the following two options are enabled 12 e start local analysis The Parafit wrapper is started with the parameters specified above e prepare data for remote analysis As an alternative all input files the Parafit wrapper including Parafit and DistPCoA and a setup file are put into a ZIP archive The archive can be transferred to a highperformance machine After archive extraction the wrapper can be invoked by the command java Xmx512M jar ParafitWrapper jar The Xmx switch denotes the maximum amount of memory the wrapper has for its own disposal here 512 MB 5 Copycat The third tab This tab see Figur
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