PRIMER v7: User Manual / Tutorial K R Clarke & R N Gorley Plymouth Routines85 In Multivariate Ecological85 Research PRIMER v7: User Manual/Tutorial K R Clarke & R N Gorley Published 2015 by PRIMER-E Ltd Business Address: 3 Meadow View, Lutton, Ivybridge, Devon PL21 9RH, United Kingdom Reproduced from 15 August 2016 by PRIMER-e (Quest Research Limited) Business Address: ecentre, Gate 5 Oaklands Rd, Massey University Albany Campus, Auckland, New Zealand First edition 2015 Clarke, K.R., Gorley, R.N. 2015 PRIMER v7: User Manual/Tutorial PRIMER-E: Plymouth © Copyright 2015, all rights reserved PRIMER v7: User Manual/Tutorial Contents OVERVIEW page A. Contact details and installation of the PRIMER v7 software Getting in touch with us 9 System requirements 9 Installing PRIMER 9 Information on analyses 9 PERMANOVA+ add-on 9 B. Introduction to the methods of PRIMER Application areas 10 Basic routines 10 C. Changes from PRIMER 6 to PRIMER 7 Wizards and major new analysis options 11 Additions to configuration (ordination) plots 13 Other new plots & plot features 14 General and miscellany 15 D. Typographic conventions for this manual Emphases and text symbols 16 Finding your way around 16 E. A brief tour through the operation of PRIMER v7 Opening the examples 17 Reading data in from Excel 17 Basic multivariate analysis (MVA) wizard 17 Pre-treatment of data 17 Matrix display wizard 18 Environmental data 18 Resemblance calculation 19 ANOSIM tests 19 CLUSTER analyses 19 MDS and PCA ordinations 19 Species analyses 20 Other analyses 20 MANUAL/TUTORIAL 0. Trial version, Help system, Manuals, Updates, Install and Uninstall (Help) PRIMER 7 trial software 21 Help system and manuals 21 Updates 22 Install and Uninstall 22 Example data 22 1. Opening, editing and saving data (File, Edit) Getting the examples 23 PRIMER file types 23 Compatibility of files 23 Opening the PRIMER 7 desktop 23 Entering data directly 24 Labelling samples and variables 24 Deleting and inserting rows/columns 25 Undo data sheet edits 25 Moving and sorting rows/columns 26 Cut, copying and pasting 26 Saving data, renaming and deleting 26 Undo in the workspace 26 Saving, closing and opening a workspace 27 Setting the initial directory 28 1 Opening PRIMER files 28 (Ekofisk oil-field fauna) 29 Properties 29 Opening Excel files 29 (Ekofisk abiotic data) 30 Wizard for input data 30 Missing or zero values? 31 (Tasmanian meiofauna) 32 Opening several files at once 32 Opening the same file twice 32 Text-format input files 32 Factors in 3-column text format files 33 Dialog for input of text format files 34 Size of data worksheets 35 Merging worksheets 35 Output data formats 35 Editing labels 36 2. Factors (and Indicators), identifying sample (and species) groups Active window 37 Use of factors 37 Creating and filling in factors 38 Cut, Copy, Paste, Delete in factors 38 Renaming and reordering factors 39 Multiple sessions and recent workspaces 39 Combining factors (e.g. to average) 40 Factor keys 41 Importing factors 42 Label matching 42 Factors in *.xls(x) or *.txt files 42 Creating indicators on variables 43 Indicators in selection 43 Variable information (aggregation files) 44 3. Highlighting and selection (Select) Highlight and select 45 (W Australia fish diets) 45 Summary statistics 45 Control of highlighting 46 Selecting and deselecting highlights 47 Duplicating a selected worksheet 47 Selecting by factor levels 47 Multiple selections 48 Selecting by number and non-missing 48 Selecting variables 49 Selecting by ‘most important’ 49 Selection in resemblance matrices 50 4. Pre-treatment options Standardising samples 51 Stats to worksheet 51 Standardising species 52 Transforming (overall) 52 Shade plots to aid choice of transform 53 Transforming abiotic variables 54 Draftsman, histogram and multi-plots 55 Transforming (individual) 56 Normalising variables 58 Dispersion weighting of species 59 (Fal estuary copepods) 60 2 Other variable weighting 61 Mixed data types 61 Variability weighting 62 (Biomarkers for N Sea flounder) 62 Cumulating samples 64 (Particle sizes for Danish sediments) 64 Surface plots 64 5. Resemblance: similarities, dissimilarities and distances Resemblance matrices 65 Standard resemblance choices 65 Bray-Curtis similarity 65 Zero-adjusted Bray-Curtis 66 (Tikus Island coral cover) 67 Euclidean distances 68 Index of Association 68 Accessing other resemblance measures 69 Distance measures 69 ‘Modified Gower’ 70 Similarity to dissimilarity 71 Quantitative similarity measures 71 Presence/Absence similarities 72 Quantitative measures on P/A data 72 Unravelling resemblances 73 Scatter plots 73 Other coefficients 74 Between-curve distances 75 (Plymouth particle-size analysis) 75 Taxonomic distinctness/aggregation files 76 Taxonomic dissimilarity measures 76 (Groundfish of European shelf waters) 77 Relatedness supplied as resemblances 77 Analysing between variables 78 Correlation between variables 79 Correlation as similarity 80 Corrections for missing data 80 Saving and opening triangular matrices 82 6. Clustering methods (CLUSTER, SIMPROF, UNCTREE, kRCLUSTER) Clustering methods and choice of linkage 83 SIMPROF tests 83 SIMPROF on large matrices 83 Modifying plots in PRIMER 83 (Exe estuary nematodes) 84 Cophenetic correlation 85 Copying and pasting plots externally 85 Sample labels & symbols menu/tab 85 Symbol and text sizes 86 Editing plot titles and scales 86 General menu/tab and Keys tab 87 Special menu for slicing and orientation of dendrograms 87 Rotating and condensing dendrograms 88 Timing bar, Stop Tasks and multi-tasking 88 Ordering factor levels in keys 89 Point and click short-cuts 89 Zooming dendrograms 89 SIMPROF method 91 (Bristol Channel zooplankton) 91 CLUSTER results window 93 3 SIMPROF direct run 93 SIMPROF Types (1-4) 94 SIMPROF on a subset of samples 94 Histograms of null distributions 95 Linkage by flexible beta method 96 Single and complete linkage 97 Limiting font size 98 Binary divisive clustering 98 UNCTREE options 99 Text pane in tree plots 99 A% and B% y-axis scales 100 Special menu for divisive trees 100 Flat-form clustering 101 7. Managing the workspace and plotting (Window, File, View, Multi Plot, Plots) Explorer tree 103 Forward and backward propagation 103 Closing, redisplaying & tiling windows 104 Minimising windows 104 View menu 104 Understanding the Explorer tree 104 Rolling up branches of the tree 105 Renaming or deleting items in a workspace 105 Undo in the Explorer tree, to reinstate or re-order 105 Saving plots 106 Vector vs. pixel plots 106 Saving graph values 107 Saving results 107 Adding notes 107 Printing results and graphs 108 Automatic creation of multi-plots 109 User creation and manipulation of multi-plots 110 Plots menu 111 Workspace planning 112 8. Multi-dimensional scaling (Non-metric nMDS, Metric mMDS, Combined MDS) Rationale for nMDS and mMDS 113 Combined MDS and ‘Fix Collapse’ 113 Diagnostic tools for MDS plots 113 Overlaying factors or other data (bubble plot) 114 Running an nMDS (Exe nematodes) 114 MDS results window 115 Shepard diagrams 115 Dissimilarity preservation as a matrix correlation 116 Accuracy and fit scheme 117 Graph menu: rotating and flipping the 2-d ordination 118 Align graphs automatically 118 Zoom and MDS subset plots 119 Special menu for ordination 120 Aspect ratio of boundary 120 Diagnostics for MDS: join pairs 120 Features that carry over to 3-d ordination 120 Minimum spanning tree (MST) 121 Linking MDS plots to cluster analysis 122 Cluster overlays on MDS plots 123 Dendrogram and 2-d MDS in a 3-d plot 124 3-d ordination plots and axes selection 124 Rotate axes or rotate and flip data 125 Drawing verticals for 3-d plots 125 4 (W Australia fish diets) 126 Higher dimensions and scree plots 126 Spinning a 3-d MDS and capture in a movie file 127 (Morlaix macrofauna, Amoco-Cadiz oil spill) 128 Overlay trajectories 128 Sequence animation, captured in 2- and 3-d 129 Trajectories split and then sequence animated 130 (Tees Bay macrofauna time series) 131 Matching variable sets 132 (Ekofisk oil-field study) 132 Bubble plots of single variables 133 Bubble colours 133 Bubble key 134 Bubble images 135 Duplicate graphs 135 Vector plots for species 136 Environment bubble and vector plots 137 Segmented bubble plots 138 (Bristol Channel zooplankton) 140 Bubble plots in 3-d MDS 140 (W Australian fish diets) 140 Bubble plot on averages 141 Bubble plot selection error and Refresh 142 Metric MDS 143 (Great circle distances for world cities) 143 Identifying points on the Shepard plot 144 Animating the mMDS and nMDS iterations 144 (Morlaix macrofauna, Amoco-Cadiz oil spill) 146 Threshold metric MDS (tmMDS) 147 Metric MDS for ordinating few points 148 ‘Fix collapse’ in nMDS 149 (Ko Phuket transects of coral reefs) 149 Combined nMDS 150 (Messolongi diatoms and abiotic data) 150 9. Analysis of Similarity tests (unordered and ordered ANOSIM) ANOSIM introduction 151 1-way layout (WA fish diet example) 152 Pairwise comparisons 153 Other 1-way ANOSIM options 154 1-way layout (Biomarkers example) 155 1-way ordered ANOSIM (Ekofisk oil-field study) 156 2-way crossed ANOSIM (Tasmanian crabs study) 158 2-way crossed ANOSIM (Danish sediment data) 159 (Phuket coral reefs) 160 1-way ordered without replication 160 2-way crossed, ordered test 161 ANOSIM for 2-way crossed design with no replication (Exe study) 163 2-way nested ANOSIM (Calafuria macroalgae) 164 3-way crossed ANOSIM (King Wrasse diets) 166 3-way fully nested design (NZ holdfast fauna) 167 3-way crossed and nested design (Tees Bay macrofauna) 169 10. Wizards and species