Plymouth Routines In Multivariate Ecological Research

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PRIMER: Getting started with v6

K R Clarke & R N Gorley

RIMER-E Ltd

Published 2005 by PRIMER-E Ltd

Registered o i!e" Ply#outh Mari$e Laboratory Pros%e!t Pla!e &est 'oe Ply#outh PL( )*' +$ited Ki$gdo# . 'edi$gha# Garde$s Roborough Ply#outh PL. /*0 +$ited Ki$gdo#

,usi$ess - i!e"

*ire!tors" K Robert Clarke M4! Ph* Ray#o$d N Gorley M5

1 Co%yright 2005 PRIMER-E Ltd2 all rights reser3ed

OVERVIEW A. Contact detai s and insta ation o! the PRIMER v6 so!tware

Getti$g i$ tou!h 7ith us 6or a$y u%-to-date $e7s about PRIMER2 i$!ludi$g details o u%!o#i$g PRIMER 7orksho%s2 see our 7eb site at" http://www.primer-e.com Please re%ort a$y bugs2 te!h$i!al %roble#s2 dislikes or suggestio$s or i#%ro3e#e$t to Ray Gorley at" tech@primer-e.com 6or li!e$si$g a$d other ge$eral e$8uiries2 !o$ta!t Cathy Clarke at" admin@primer-e.com +se this latter e-#ail address to !o$ta!t ,ob Clarke2 or 8ueries related to the s!ie$ti i! #ethods9 -ur busi$ess %ostal address is" PRIMER-E Ltd . 'edi$gha# Garde$s Roborough Ply#outh PL. /*0 +$ited Ki$gdo# Tel/Fax: +44 ( !"#$% #&''(( :or you #ay2 i you %re er2 use the registered address o the !o#%a$y" PRIMER-E Ltd2 Ply#outh Mari$e Laboratory2 Pros%e!t Pla!e2 &est 'oe2 Ply#outh PL( )*'2 +$ited Ki$gdo#; 4yste# re8uire#e$ts PC 7ith I$tel !o#%atible %ro!essor &i$do7s <=>2000>Me>N?@>0P %ossible2 but &i$do7s 20002 0P or later re!o##e$ded Me#ory9 6or &i$do7s <= a$d Me" .@ Mb :but at least (2= Mb re!o##e$ded;9 6or &i$do7s 20002 0P or later" (2= Mb :25. Mb re!o##e$ded; I$ter$et EA%lorer 595 or later is $eeded to i$stall the 9Net e$3iro$#e$t ?o be able to read a$d 7rite EA!el 7orksheets you $eed EA!el 20002 or later2 i$stalled o$ the PC ?his is a sta$d-alo$e %rodu!t or i$stallatio$ o$ a$ i$di3idual PC2 $ot a $et7ork 3ersio$9 Bou $eed to be logged o$ as 5d#i$istrator or &i$N?2 20002 0P or later9 I you ha3e a$ earlier PRIMER 3. i$stalled :i$!ludi$g a beta-test 3ersio$;2 you irst $eed to u$i$stall it2 usi$g 5dd or Re#o3e Progra#s ro# the Co$trol Pa$el9 Bou do $ot $eed to u$i$stall PRIMER 35 :or earlier 3ersio$s;9 I$sert the PRIMER C* i$ your C* dri3e9 ?he i$stall %rogra# 7ill auto#ati!ally ru$ u$less you ha3e disabled autoru$ o$ your dri3e9 :I it does $ot ru$2 o%e$ &i$do7s EA%lorer2 sele!t the C* dri3e a$d double !li!k o$ 4etu%9eAe9; ?he i$stall %rogra# 7ill i$stall the 9Net e$3iro$#e$t o$ your !o#%uter i it is $ot already u% to date9 Bou 7ill be asked or your serial $u#ber a$d key 7hi!h is o$ the ro$t o the C* !ase9 4etu% #ay reboot your syste# duri$g the i$stallatio$ a$d2 as usual2 it is ad3isable to !lose do7$ all other %rogra#s be ore !o##e$!i$g i$stallatio$9 Getti$g hel% Ge$eral i$ or#atio$ about the te!h$i8ues u$derlyi$g the a$alyses are ou$d i$ the a!!o#%a$yi$g Methods #a$ual" CCha$ge i$ Mari$e Co##u$itiesD2 by Clarke2 &ar7i!k2 4o#er ield a$d Gorley :2005;9 I$ or#atio$ s%e!i i! to PRIMER . is !o$tai$ed i$ a +ser #a$ual>?utorial a$d the so t7are 'el% syste#9 ?he latter is !o$teAt se$siti3e" i you !li!k o$ the 'el% butto$ i$ a dialog the$ you 7ill get a$ a%%ro%riate hel% to%i!9 Bou !a$ also get i$to the hel% syste# by !hoosi$g the 'el%ECo$te$ts #e$u o%tio$ or !li!ki$g o$ the hel% butto$ o$ the butto$ bar9 Bou !a$ the$ bro7se this syste# 3ia the Co$te$ts or I$deA tabs i$ the 'el% 7i$do79 I you are still ha3i$g %roble#s2 !o$ta!t the sta at PRIMER-E :see the to% o this %age;2 7ho 7ill be ha%%y to hel%9

I$stalli$g PRIMER

". Introd#ction to the $ethods o! PRIMER 5%%li!atio$ areas PRIMER . :Ply#outh Routi$es I$ Multi3ariate E!ologi!al Resear!h; !o$sists %ri#arily o a 7ide ra$ge o u$i3ariate2 gra%hi!al a$d #ulti3ariate routi$es or a$alysi$g arrays o s%e!ies-by-sa#%les data ro# !o##u$ity e!ology9 *ata are ty%i!ally o abu$da$!e2 bio#ass2 F !o3er2 %rese$!e> abse$!e et!2 a$d arise i$ biologi!al #o$itori$g o e$3iro$#e$tal i#%a!t a$d #ore u$da#e$tal studies2 e9g9 o dietary !o#%ositio$9 5lso !atered or are #atri!es o %hysi!al 3alues a$d !he#i!al !o$!e$tratio$s2 7hi!h are a$alysed i$ their o7$ right or i$ %arallel 7ith biologi!al asse#blage data2 CeA%lai$i$gD !o##u$ity stru!ture by %hysi!o-!he#i!al !o$ditio$s9 ?he #ethods o this %a!kage #ake e72 i a$y2 assu#%tio$s about the or# o the data :G$o$-#etri!G ordi$atio$ a$d %er#utatio$ tests are u$da#e$tal to the a%%roa!h; a$d !o$!e$trate o$ a%%roa!hes that are straight or7ard to u$dersta$d a$d eA%lai$9 ?his robust$ess #akes the# 7idely a%%li!able2 leadi$g to greater !o$ ide$!e i$ i$ter%retatio$2 a$d the tra$s%are$!y %ossibly eA%lai$s 7hy they ha3e bee$ ado%ted 7orld7ide2 %arti!ularly i$ #ari$e s!ie$!e but i$!reasi$gly i$ terrestrial2 resh7ater2 %aleo$tology et! !o$teAts9 ?he statisti!al #ethods u$derlyi$g the so t7are are eA%lai$ed i$ $o$-#athe#ati!al ter#s i$ the asso!iated C#ethodsD #a$ual :Clarke et al 2005;2 7hi!h also sho7s out!o#es ro# #a$y literature studies2 e9g9 o e$3iro$#e$tal e e!ts o oil s%ills2 drilli$g #ud dis%osal a$d se7age %ollutio$ o$ so t-sedi#e$t be$thi! asse#blages2 disturba$!e or !li#ati! e e!ts o$ !oral ree !o#%ositio$ or ish !o##u$ities2 #ore u$da#e$tal biodi3ersity a$d !o##u$ity e!ology %atter$s2 #eso!os# studies 7ith #ulti-s%e!ies out!o#es et!9 Ma$y o these ull data sets are i$!luded 7ith the %a!kage so that the user !a$ re%li!ate the a$alyses gi3e$ i$ the #a$ual9 ?hough the a$alysis re8uire#e$ts or biologi!al asse#blage data are a %ri$!i%al o!us2 the %a!kage is e8ually a%%li!able :a$d i$!reasi$gly bei$g a%%lied; to other data stru!tures 7hi!h are either #ulti3ariate or !a$ be treated as su!h9 ?hese i$!lude" #ulti%le bio#arkers i$ e!otoAi!ology2 a$d their relatio$ to 7ater or tissue !o$!e$tratio$s o !he#i!al !o$ta#i$a$tsH !o#%ositio$ o substrate i$ geology or #aterials s!ie$!eH #or%ho#etri! #easure#e$ts i$ taAo$o#i! dis!ri#i$atio$H ge$eti! studies2 i$3ol3i$g %rese$!e or abse$!e o s%e!i i! sets o allelesH sig$als at #ulti%le 7a3ele$gths i$ re#ote se$si$g2 !hara!terisi$g 3egetatio$ or 7ater #asses2 et!9 +$i3ariate #easure#e$ts 7hi!h !a$ so#eti#es be treated #ore e e!ti3ely i$ #ulti3ariate ashio$ i$!lude %arti!le siIe a$alysis or 7ater or sedi#e$t sa#%les a$d siIe re8ue$!y distributio$s o orga$is#s i$ !ohort studies :the #ulti3ariate 3ariables are the dis!rete %arti!le or orga$is# siIe !lasses;9 4ets o gro7th !ur3es or i$di3idual orga$is#s2 #o$itored through ti#e :Cre%eated #easuresD; are also readily a$alysed" the u$i yi$g eature is that all data sets are redu!ed to a$ a%%ro%riate tria$gular #atriA re%rese$ti$g the :dis;si#ilarity o e3ery %air o sa#%les2 i$ ter#s o their asse#blages2 suites o bio#arkers2 %arti!le siIe distributio$s2 sha%e o gro7th !ur3es2 et!9 Clusteri$g a$d ordi$atio$ te!h$i8ues are the$ able to re%rese$t the relatio$shi%s bet7ee$ the sa#%les2 a$d %er#utatio$ tests i#%ose a $e!essary hy%othesis testi$g stru!ture9 ?o de#o$strate the ra$ge o a%%li!atio$ areas2 re ere$!es to I4I-listed %ubli!atio$s 7hi!h !ite earlier 3ersio$s o the PRIMER #ethods #a$ual :a$d o$e o the !ore #ethods %a%ers2 Clarke (<<); !a$ be do7$load ro# the PRIMER-E 7ebsite :7779%ri#er-e9!o#;9 ,asi! routi$es ?he basi! routi$es o the %a!kage !o3er" hierar!hi!al !lusteri$g i$to sa#%le :or s%e!ies; grou%s :CL+4?ER;H ordi$atio$ by $o$-#etri! #ultidi#e$sio$al s!ali$g :M*4; a$d %ri$!i%al !o#%o$e$ts :PC5; to su##arise %atter$s i$ s%e!ies !o#%ositio$ a$d e$3iro$#e$tal 3ariablesH %er#utatio$based hy%othesis testi$g :5N-4IM;2 a$ a$alogue o u$i3ariate 5N-J5 7hi!h tests or di ere$!es bet7ee$ grou%s o :#ulti3ariate; sa#%les ro# di ere$t ti#es2 lo!atio$s2 eA%eri#e$tal treat#e$ts et!H ide$ti yi$g the s%e!ies %ri#arily %ro3idi$g the dis!ri#i$atio$ bet7ee$ t7o obser3ed sa#%le !lusters :4IMPER;H the li$ki$g o #ulti3ariate bioti! %atter$s to suites o e$3iro$#e$tal 3ariables or other bioti! arrays :,E4? a$d LINK?REE;H !o#%arati3e :Ma$tel-ty%e; tests o$ si#ilarity #atri!es :REL5?E;H a se!o$d-stage M*4 routi$e i$ 7hi!h relatio$shi%s bet7ee$ a large set o ordi$atio$s !a$ be 3isualised :24?5GE;H sta$dard di3ersity i$di!esH do#i$a$!e %lotsH geo#etri! abu$da$!e distributio$sH s%e!ies a!!u#ulatio$ esti#atorsH aggregatio$ o arrays to allo7 data a$alysis at higher taAo$o#i! le3els2 et!9 5 urther u$i8ue eature o PRIMER is the ability to !al!ulate a$d test biodi3ersity i$di!es based o$ the taAo$o#i! disti$!t$ess o the s%e!ies #aki$g u% a 8ua$titati3e sa#%le or s%e!ies list2 i$di!es 7hose statisti!al %ro%erties are robust to 3aryi$g ri!h$ess9 ?hese %er#it testi$g or !ha$ge i$ biodi3ersity :?50*?E4?;2 by !o#%ariso$ 7ith a regio$al Cs%e!ies %oolD2 so that di3ersity %atter$s o3er 7ide s%a!e>ti#e s!ales are !o#%arable 7he$ sa#%li$g e ort is u$!o$trolled 9
2

C. Changes !ro$ PRIMER 5 to PRIMER 6 ?e!h$i!al (; Mo3ed e$tirely i$to the $e7 Mi!roso t 9NE? e$3iro$#e$t2 gi3i$g the so t7are a ully #oder$ &i$do7s a%%eara$!e2 sa eguardi$g uture gro7th %aths a$d o%e$i$g the 7ay to di3ersi i!atio$ o$to di ere$t %lat or#s :+NI02 Ma!; i$ uture9 :Curre$tly 3. is o$ly a3ailable i$ a &i$do7s e$3iro$#e$t2 though this !ould be a Jirtual &i$do7s e$3iro$#e$t o$ a Ma!2 or eAa#%le;9 2; Re%la!e#e$t o i$e i!ie$t a$d i$ leAible gra%hi!s a$d grid !o$trols :e9g9 Gra%hi!s 4er3er;2 by $ati3e !ode i$ 9NE?2 has greatly i#%ro3ed Clook a$d eelD o the gra%hi!s2 7hi!h are $o7 ully tailored to the $eeds9 ?his has also !ut through s%eed bottle$e!ks i$ ha$dli$g large s%readsheets9 ); No iAed siIe !o$strai$ts o$ data #atri!es or grou% siIes or a$y a$alysis9 ?he li#itatio$s are i#%osed o$ly by total a3ailable R5M9 &i$do7s 2000 or later2 a$d 25.Mb R5M2 re!o##e$ded9 No lo$ger %ossible to ru$ o$ &i$do7s <59 @; 4%eed gai$s o arou$d a a!tor o 5 i$ #ost o the hea3ily-!o#%utatio$al algorith#s :M*42 ,I--ENJ sear!hes2 %er#utatio$ tests et!;H larger s%eed gai$s i$ #a$i%ulati$g data 7i$do7s2 es%e!ially or 3ery large data sets :e9g9 o%e$i$g a$d !losi$g a!tor 7i$do7s;9 5; No7 ully #ulti-taski$g9 Bou !a$ start se3eral 3ery lo$g a$alyses a$d !o$ti$ue doi$g other thi$gs i$ the 7orks%a!e9 ?his takes ull ad3a$tage o #ulti-%ro!essor syste#s :di ere$t tasks 7ill auto#ati!ally be allo!ated to the least loaded %ro!essor;9 I$ter a!e .; ?he 7orks%a!e $o7 dis%lays a$ CEA%lorer treeD 7hi!h !a$ be $a3igated to re!all i$sta$tly a$y o the deri3ed 7orksheets2 %lots or results 7i$do7s9 ?his is $ot solely a dis%lay" the tree stru!ture is used i$ter$ally to %ass i$ or#atio$ bet7ee$ related data sheets or %lots :e9g9 $e7 a!tors de i$ed ro# a CL+4?ER de$drogra# are ba!k-%ro%agated through the si#ilarity #atriA to the origi$al data #atriA2 a$d or7ard-%ro%agated to a$ eAisti$g M*4 %lot ro# that sa#e si#ilarity sheet;9 Results 7i$do7s are $o7 lo!al to ea!h a$alysis2 #aki$g it easier to i$d results at a later stage9 /; &orks%a!es are $o7 sa3eable i$ their e$tiretyH PRIMER 3. !a$ be shut do7$ a$d re-o%e$ed at a later date2 re!alli$g the 7orks%a!e !o$te$t i$ eAa!tly the or# it 7as le t :i9e9 irres%e!ti3e o a$y subse8ue$t !ha$ges or deletio$s to the data iles that 7ere origi$ally read-i$ to the 7orks%a!e;9 =; I#%ro3ed data e$try dialog a$d 7ider sele!tio$ o i$%ut or#ats9 Re!ta$gular EA!el sheets o 3ariables by sa#%les :or sa#%les by 3ariables; are read i$ easily usi$g a $e7 e$try C&iIardD2 7hi!h $o7 allo7s !hoi!e o sheet 7ithi$ the EA!el ile by $a#e9 PRIMER does $ot ha3e the EA!el !o$strai$t to 255 !olu#$s9 Larger iles !a$ be read-i$ ro# #ulti%le EA!el sheets a$d Merged or2 i !reated by a database %a!kage i$ three-!olu#$ or#at :Csa#%le $u#ber2 3ariable $u#ber2 3alueD;2 !a$ $o7 be read dire!tly i$to PRIMER K a$d 7ill be auto#ati!ally !o$3erted to re!ta$gular or#at9 <; ?here is $o7 label #at!hi$g" sa#%les or 3ariables do $ot $eed to a%%ear i$ the sa#e order or those a$alyses that #at!h t7o di ere$t sheets :e9g9 ,I--ENJ2 REL5?E2 5,C do#i$a$!e %lots2 5ggregatio$ et!;9 Bou usually o$ly $eed to 7orry about the sele!tio$ i$ the a!ti3e 7orksheet a$d 3. 7ill %er or# a$y sele!tio$ or re-orderi$g $eeded or other sheets9 -$e #aLor ad3a$tage o label #at!hi$g is the auto#ati! #ergi$g o t7o s%e!ies-by-sa#%les sheets i$ 7hi!h s%e!ies lists are o$ly %artly o3erla%%i$g :a$d i$ a di ere$t order;9 Co$siste$t s%elli$g o labels is re8uired2 $aturallyM (0; &orksheets are gi3e$ a$ eA%li!it data ty%e :abu$da$!e2 bio#ass2 e$3iro$#e$t2 other;2 7hi!h %er#it se$sible de aults a$d 7ar$i$gs :e9g9 i 5,C i$%uts are $ot o ty%es abu$da$!e a$d bio#ass;9 ((; Ne7 data ha$dli$g o%eratio$s i$!lude" ra$ki$g o 3ariables :e9g9 a$ alter$ati3e to i$di3idually tra$s or#i$g e$3iro$#e$tal 3ariables;H sorti$g :by $a#e or a!tor le3els; a$d #o3i$g dataH #ore %o7er ul i$di3idual tra$s or# o%tio$s2 that !a$ $o7 !o#bi$e di ere$t sa#%les2 3ariables2 $u#eri! a!tors a$d i$di!ators2 as 7ell as other 7orksheets :usi$g the $e7 label #at!hi$g;H #ore eAte$si3e !he!ks o$ i$%ut data :i$!ludi$g 3alidatio$ o aggregatio$ iles;H a$d a $e7 4u# tool9 (2; I#%ro3ed out%ut or#ats2 a$d a 7ider ra$ge o o%tio$s9 ?ables i$ the results 7i$do7s are or#atted to be able to !o%y a$d %aste dire!tly i$to EA!el s%readsheets9 Ma$y routi$es 7ill $o7 allo7 su##ary i$ or#atio$ to be se$t to a $e7 7orksheet : or urther o%eratio$ or eA%ort; i$stead o to teAt- or#at results9 ?here is a #ulti%le-%age %ri$t o%tio$2 use ul or large de$drogra#s2 a$d additio$al gra%hi!s out%ut or#ats2 i$!ludi$g 9L%g2 9ti 2 9%$g9 ?he sta$dard 3e!tor or#at is the &i$do7s e$ha$!ed #eta ile :9e# ;9 6i$al %oi$t !o-ordi$ates :e9g9 o the $e7 %ositio$ o sa#%les a ter rotatio$ o a$ M*4 %lot; !a$ also be out%ut9
)

(); *ata sheets $o7 %er#it a!tors o$ the sa#%les :or i$di!ators o$ the 3ariables; to be read i$ ro# :or sa3ed to; the EA!el or teAt or#ats2 allo7i$g ra%id tra$s er o all i$ or#atio$ bet7ee$ or#ats9 6a!tors are $o7 easily i#%orted ro# other data sheets2 !o#bi$ed to %rodu!e !o#%osite le3els :e9g9 a site-by-ti#e !o#%osite a!tor !ould be used to 53erage or 4u# o3er re%li!ates or ea!h site>ti#e le3el;2 a$d le3els !a$ be gi3e$ de ault sy#bols a$d !olours or use i$ all %lots9 (@; I#%ro3ed ti#er2 sho7i$g ho7 lo$g a %arti!ular a$alysis has le t to ru$2 %lus a 4to% butto$2 so that a$y a$alysis 7hi!h threate$s to take too lo$g to eAe!ute !a$ be s7i tly a$d !lea$ly ter#i$ated9 E$ha$!ed gra%hi!s (5; I$ ordi$atio$ %lots :M*42 PC5;2 #ore leAible additio$ o sy#bols a$d labels de$oti$g :di ere$t; a!tors9 ?he )-d %lotti$g routi$e is greatly i#%ro3ed2 both i$ res%e!t o rotatio$ o aAes :a$d %oi$ts2 or M*4; a$d re le!tio$ o aAes2 7ith the ability to add labels to %oi$ts i$ additio$ to sy#bols9 I$ or#atio$ dis%layed o$ %lots is #ore !o#%rehe$si3e2 7ith the auto#ati! use o subtitles a$d a Chistory boAD to allo7 !lear di ere$tiatio$ bet7ee$ %lots ro# di ere$t tra$s or# a$d si#ilarity o%tio$s2 a$d a key or bubble siIe is gi3e$ 7he$ a 3ariable is su%eri#%osed9 (.; I$ !lusteri$g2 keyed sy#bols !a$ $o7 be added :i$de%e$de$tly o labels; to de$drogra#s2 a$d the latter !a$ be dis%layed i$ a$y o$e o our orie$tatio$s9 Clusters %rodu!ed at a iAed si#ilarity le3el !a$ be ide$ti ied a$d sa3ed as a a!tor2 a$d !a$ the$ be dis%layed o$ the #at!hi$g M*49 5lter$ati3ely2 a$ M*4 %lot !a$ sho7 the results o a !luster a$alysis as s#oothed !o$3eA !o$tours2 dra7$ rou$d %oi$ts that are !lustered at se3eral s%e!i ied rese#bla$!e le3els9 (/; Poi$ts i$ a$ ordi$atio$ :i$ 2- or )-d; !a$ be Loi$ed by straight li$e seg#e$ts2 i$ a de i$ed order2 allo7i$g tre$ds i$ ti#e2 s%a!e or e$3iro$#e$tal gradie$t to be #ore readily see$9 (=; M*4 diag$osti!s are i#%ro3ed" 4he%hard %lots o dista$!es i$ the ordi$atio$ agai$st origi$al dissi#ilarities are gi3e$ or both 2- a$d )-d solutio$sH F !o$tributio$s o i$di3idual %oi$ts to the stress le3el are listedH a$ alter$ati3e itti$g s!he#e allo7s a di ere$t treat#e$t o tied si#ilarities2 a$d the user has #ore !o$trol o3er the %re!isio$ o out%ut stress 3alues9 (<; I$ PC52 3e!tor %lots are $o7 %er#itted :si$!e the aAes are li$ear !o#bi$atio$s o the 3ariables; a$d a$y %air or tri%le o PCs !a$ be %lotted i$ the 2- or )-d !o$ iguratio$s9 20; *o#i$a$!e %lots ro# a si$gle sheet :abu$da$!e or bio#ass; !a$ ha3e the #ulti%le li$es ide$ti ied as di ere$t le3els o a a!tor :by di eri$g li$e !olours a$d>or sy#bols;9 6or 5,C !ur3es2 the ull set o %lots ro# all sa#%les are $o7 auto#ati!ally !reated i$ o$e ru$ o the routi$e9 ?he :!o$ usi$g; o%tio$ i$ 35 o auto#ati! su##atio$ o sele!ted sa#%les has bee$ re#o3ed :i $eeded2 it !a$ be !arried out usi$g the $e7 4u# tool %rior to e$teri$g *o#i$a$!e or Geo#etri! %lot #e$u;9 2(; ?he C u$$elD a$d Celli%seD %lots or a3erage :a$d 3aria$!e i$; taAo$o#i! disti$!t$ess i$di!es are also e$ha$!ed2 7ith #ore leAible !hoi!e o labels a$d sy#bols2 !olour shadi$g et! 22; Perha%s the #ost use ul e$ha$!e#e$t is the ability to Ioo# i$ o$ the !o$te$t o a$y %ortio$ o a %lot :ordi$atio$2 de$drogra#2 u$$el %lot2 dra ts#a$ %lot et!;2 7hilst retai$i$g aAes a$d s!ales2 a$d ha3i$g the ability to s!roll through the %lot at the !urre$t #ag$i i!atio$9 ?his e3e$ eAte$ds to !ha$gi$g the as%e!t ratio :though $ot or ordi$atio$sM;2 by irst dra7i$g a re!ta$gle arou$d the %ortio$ to #ag$i y9 ?his is %arti!ularly use ul or de$drogra#s2 7hi!h or large $u#bers o %oi$ts $eed to #ag$i y the sa#%le s!ale but $ot the si#ilarity s!ale9 2); 6or M*4 ordi$atio$s2 i$terest is usually less i$ #ag$i yi$g a subset o the %lot tha$ re%eati$g the ordi$atio$ Lust or that subset o %oi$ts" agai$2 dra7i$g a boA arou$d the subset o$ the %lot allo7s a re-ru$ o the M*4 or those %oi$ts2 7ith a si$gle !li!k o$ a #e$u>toolbar i!o$M 5dditio$al a$alyses 2@; Pre-treat#e$t o%eratio$s are greatly eA%a$ded" a; sta$dardisatio$ o%tio$s are $o7 by s%e!ies or 3ariable2 a$d by total or #aAi#u#H b; a !u#ulate o%eratio$ assists i$ 7ide$i$g the a%%li!atio$ o these routi$es to siIe !lasses i$ %arti!le or body-siIe distributio$sH !; 3ariables !a$ be 7eighted2 e9g9 do7$7eighti$g so#e s%e!ies o$ the basis o #iside$ti i!atio$ rates9 -3erall tra$s or#atio$ a$d $or#alisatio$ o%tio$s re#ai$ as i$ 35 but2 i#%orta$tly2 all %re-treat#e$ts $o7 be!o#e a$ i$itial ste%2 se%arated ro# si#ilarity !al!ulatio$s2 allo7i$g a highly leAible ra$ge o alter$ati3es9

25; ?he $e7 user is still guided to Csa eD2 sta$dard !hoi!es :e9g9 ,ray-Curtis o$ tra$s or#ed s%e!ies abu$da$!es2 $or#alised Eu!lidea$ dista$!e or e$3iro$#e$tal 3ariables;2 but the #ore eA%erie$!ed user $o7 has a!!ess to $early 50 si#ilarity>dissi#ilarity>dista$!e #easures2 dra7$ ro# di ere$t literature !o$teAts2 a$d !olle!ti3ely re erred to as Crese#bla$!e #easuresD9 2.; Ne7 resear!h te!h$i8ues here are" a; Cdis%ersio$ 7eighti$gD2 the do7$7eighti$g o i$di3idual s%e!ies !ou$ts usi$g their i$$ate heteroge$eity i$ re%li!ates2 arisi$g ro# !lustered s%atio-te#%oral distributio$sH b; CtaAo$o#i! dissi#ilaritiesD2 eAte$di$g the !o$!e%ts o taAo$o#i! disti$!t$ess i$trodu!ed i$ PRIMER 35 ro# di3ersity #easures to dissi#ilarity !oe i!ie$ts9 2/; Missi$g 3alues are $o7 tolerated i$ so#e routi$es a$d2 7here they are s%arse2 !a$ be assu#ed C#issi$g at ra$do#D2 #a$y e7er 3ariables tha$ sa#%les are sele!ted2 a$d the 3ariables !a$ be tra$s or#ed to a%%roAi#ate $or#ality :e9g9 e$3iro$#e$tal-ty%e 3ariables;2 a$ EM :EA%e!tatio$MaAi#isatio$; algorith# or o%ti#al %redi!tio$ o #issi$g data is %ro3ided9 :+se 7ith !areM; 2=; *ata ha$dli$g o%eratio$s su!h as tra$s or#i$g a$d ra$ki$g $o7 a%%ly to rese#bla$!e #atri!es2 7hi!h allo7s !o$3ersio$ o !orrelatio$ #atri!es to si#ilarities2 e9g9 to obtai$ e$3iro$#e$tal 3ariable M*4 %lots9 4i#ilarities !a$ be eA%li!itly s7it!hed to dissi#ilarities2 a$d 3i!e-3ersa2 a$d a$other rese#bla$!e tool is a CModel #atriAD2 or buildi$g #odel #atri!es i$ REL5?E tests :$o$%ara#etri! Ma$tel-ty%e;" these #ight re%rese$t serial or !y!li! tre$ds 7ith re%li!atio$ o grou%s9 2<; ?he si#ilarity %er!e$tages routi$e :4IMPER;2 ide$ti yi$g 3ariables that !o$tribute #ost to the di ere$!e bet7ee$ t7o grou%s2 is eA%a$ded to !o3er t7o- a!tor desig$s a$d to a%%ly to Eu!lidea$ dista$!e :e9g9 or e$3iro$#e$tal 3ariables; as 7ell as ,ray-Curtis9 -ther #e$u ite#s are regrou%ed" ,I--ENJ a$d ,J4?EP are #erged :i$to ,E4?; a$d 5N-4IM2 absorbed i$to the 5N-4IM #e$uH all global %er#utatio$ tests $o7 !reate histogra#s o $ull distributio$s9 )0; 4e3eral $e7 global sig$i i!a$!e tests are %ro3ided :robust2 $o$-%ara#etri!2 %er#utatio$-based a$d eAa!t;2 to test or %rese$!e o #ea$i$g ul stru!ture be ore i$ter%reti$g results a$d dis%lays" a; *-M*I42 7hi!h !o#%utes dista$!es bet7ee$ the !ur3es i$ do#i$a$!e %lots2 7hi!h !a$ the$ be i$%ut to a$ 5N-4IM test o a priori grou% stru!tureH b; a ,I--ENJ test2 7hi!h allo7s or the bias i$here$t i$ o%ti#al sele!tio$ o a subset o 3ariables ro# o$e #atriA :e9g9 e$3iro$#e$tal; that best CeA%lai$sD the %atter$ i$ a se!o$d :e9g9 s%e!ies;H !; the 4IMPR-6 :si#ilarity %ro ile; routi$e2 7hi!h tests or e3ide$!e o stru!ture i$ a$ a priori u$stru!tured set o sa#%les9 I$ !o#bi$atio$ 7ith !lusteri$g a$d a $e7 a!ility to !o$de$se s%e!i i! substru!tures i$ de$drogra#s :by !li!ki$g o$ 3erti!al li$es;2 this !a$ ge$erate trees that are %ru$ed to obLe!ti3ely-de i$ed grou%s9 5 a!tor de i$i$g these grou%s !a$ be auto#ati!ally !reated9 )(; LINK?REE2 a $e7 routi$e to li$k bioti! %atter$s to e$3iro$#e$tal 3ariables :or si#ilar li$kage %roble#s;9 Li$kage trees K a $o$-%ara#etri! 3ersio$ o #ulti3ariate regressio$ trees or C5R?s K o%ti#ise su!!essi3e bi$ary di3isio$s o bioti! %atter$s usi$g threshold 3alues o e$3iro$#e$tal 3ariables2 a$d ai# to %ro3ide %ie!e#eal CeA%la$atio$sD o asse#blage grou%i$gs i$ ter#s o si$gle e$3iro$#e$tal 3ariables9 It is e e!ti3e i$ !o$Lu$!tio$ 7ith the ,I--ENJ :,E4?; routi$e2 or #ay %ro3ide better eA%la$atio$s 7here ,I--ENJ ails be!ause or!i$g 3ariables are 3ery $o$-additi3e9 It !a$ be !o#bi$ed 7ith 4IMPR-62 to test or the Csig$i i!a$tD stru!ture re8uiri$g eA%la$atio$9 )2; 4%e!ies-a!!u#ulatio$ %lots $o7 !al!ulate a$d %lot a 7ide ra$ge o ri!h$ess esti#ators :Chao (&22 Na!k$i e (&22 ,ootstra%2 Mi!haelis-Me$te$2 +GE; as sa#%les are !u#ulated i$ origi$al2 s%e!i ied or %er#uted orders9 Results are se$t to a 7orksheet or easy eA%ort9 )); 5 u$i8ue eature o 35 7as the !al!ulatio$ o taAo$o#i! disti$!t$ess #easures o biodi3ersity2 based o$ taAo$o#i! or %hyloge$eti! related$ess o s%e!ies i$ a sa#%le2 a$d testi$g or Cbiodi3ersity lossD by si#ulati$g sele!tio$ ro# a #aster s%e!ies list :?50*?E4?;9 3. e$ha$!es this $ot o$ly by better gra%hi!s but by the ability to use a di ere$t #aster list or the si#ulatio$ tha$ the sa#%le data :e9g9 ossil lists !a$ $o7 be !o#%ared 7ith #oder$ o$es;2 a$d the o%tio$ to s%e!i y a data sheet that deter#i$es the re8ue$!y 7ith 7hi!h ea!h s%e!ies is dra7$ ro# the #aster s%e!ies list2 7he$ !o$stru!ti$g the C$ull #odelD si#ulatio$s9

%. &'(ogra(hic conventions
?eAt i$ )o d i$di!ates the #e$u ite#s that $eed to be sele!ted2 * de$otes !as!adi$g sub-#e$u ite#s2 tab !hoi!es2 dialog boAes or sub-boAes2 de$otes a butto$ e$try i$ a dialog boA :so-!alled Cradio butto$sD K o$ly o$e !a$ be sele!ted;2 i$di!ates a ti!k i$ the s%e!i ied boA :so !alled C!he!k boAesD K either o$ or o ;2 teAt i$side a !artou!he is a$ i$stru!tio$ to sele!t the suggested e$try :e9g9 ile$a#e; or a!tually to ty%e it i$2 a$d : ; & : ; & : ; i$di!ate se3eral ste%s that $eed to be !arried out i$ the o$e boA2 7here bra!kets are used $aturally to s%lit u% the di ere$t !o#%o$e$ts o the dialog9 6or eAa#%le2 Ana 'se*Rese$) ance*:5$alyse bet7ee$4a#%les; & :Measure,ray-Curtis si#ilarity; & :5dd du##y 3ariableEJalue" (; is a$ i$stru!tio$ to sele!t the #ai$ #e$u ite# 5$alyse2 the sub-#e$u ite# Rese#bla$!e2 a$d a$alyse bet7ee$ sa#%les usi$g ,ray-Curtis si#ilarity2 addi$g a du##y s%e!ies 7ith 3alue ( or all sa#%les2 %rior to !o#%uti$g si#ilarities :this is a #odi i!atio$ to the ,ray-Curtis !oe i!ie$t to or!e it to beha3e i$ a %arti!ular 7ay as the sa#%les be!o#e 3ery s%arse K see the Methods #a$ual or urther dis!ussio$ o the e e!t o this adLust#e$t;9 ?he dialog this !orres%o$ds to is"

E. A )rie! to#r thro#gh the o(eration o! PRIMER v6

Readi$g i$ the eAa#%les ?he irst ste% is to get data i$to PRIMER9 *ata is held i$ 7orksheetsH you !a$ read i$ data iles i$ se3eral or#ats2 or you !a$ e$ter a$d edit data dire!tly9 ?he PRIMER C* !o$tai$s a $u#ber o eAa#%le data iles" i $e!essary2 !o%y the EAa#%les 3. dire!tory ro# the C* to your hard dri3e9 :I you i$stalled to the de ault lo!atio$ you should i$d the EAa#%les 3. dire!tory already u$der C"OProgra# 6ilesOPRIMER-EOPRIMER .OEAa#%les 3.9; ?o read i$ data2 sele!t +i e*O(en ro# the #ai$ #e$u a$d $a3igate to2 or eAa#%le2 the EAa#%les 3.OEko isk subdire!tory2 sele!ti$g the ek#a9%ri ile9 ?his is o #ari$e s%e!ies asse#blages at sites rou$d a$ oil ield drilli$g rig :see the des!ri%tio$ boA ro# Edit*Pro(erties;9 ?he data !a$ be i$%ut either as sa#%les :ro7s; by 3ariables :!olu#$s;2 7hi!h is the !o$3e$tio$ i$ statisti!s2 or as 3ariables by sa#%les2 the !o$3e$tio$ or biologi!al #atri!es2 but PRIMER does $eed to k$o7 7hi!h are the sa#%les a$d 7hi!h the 3ariablesM Che!k this ro# the Edit*Pro(erties #e$u 7here :4a#%les asColu#$s; is the !orre!t !hoi!e here9 5s 7ell as its o7$ 3. or#at :9%ri eAte$sio$;2 PRIMER !a$ dire!tly read i$ teAt iles2 EA!el iles a$d the old PRIMER data or#ats9 Che!k the e$3iro$#e$tal data ile or Eko isk2 i$ Eko iskO eke39Als2 by o%e$i$g it i$ EA!el2 to see the re!ta$gular or#at that PRIMER eA%e!ts9 Note that it ha%%e$s to be tra$s%osed :sa#%les by 3ariables; !o#%ared 7ith the s%e!ies #atriA9 ?he e$tries #ust be $u#eri!2 though ro7 a$d !olu#$ labels !a$ be al%ha$u#eri!9 ?he sa#%le labels are u$i8ue a$d #at!h those or the biologi!al data2 7hi!h 7ill allo7 !o#%ariso$ o the t7o arrays2 though the sa#%les $eed $ot be i$ the sa#e order i$ the t7o #atri!es :a !ha$ge ro# 35;9 Larger #atri!es tha$ !a$ be ha$dled by EA!el i$ re!ta$gular or#at :i9e9 E255 !olu#$s;2 !a$ be read i$ as )-!olu#$ teAt iles :Csa#%le label2 3ariable label2 3alueD o$ ea!h li$e2 7ith tab2 !o##a or other s%e!i ied se%arator;2 or s#aller sub-#atri!es read i$ se%arately a$d #erged i$ PRIMER9 4ele!t +i e*O(en ro# the #e$us9 Bou !a$ sele!t the ile ty%es to read usi$g the boA at the botto# o the dialog9 ?he de ault setti$g 7ill read PRIMER 5 :a$d PRIMER @; iles as 7ell as the $e7 PRIMER . or#at2 but you ha3e to sele!t the EA!el o%tio$ to dis%lay the P9Als iles9 Go through the ste%s i$ readi$g i$ this %arti!ular EA!el ile2 by +i e*O(en*:6ile $a#e" eke39Als; & :6iles o ty%e" EA!el iles :P9Als;;EO(en2 the$ i$ the EA!el 6ile &iIard take all the de aults eA!e%t :-rie$tatio$4a#%les as ro7s; & :*ata ty%e E$3iro$#e$tal; o$ the se!o$d dialog boA9 E$teri$g data dire!tly i$ 3. ?o i$%ut data dire!tly i$to PRIMER !hoose +i e*,ew. 6or 7orksheet ty%e !hoose 4a#%le data a$d i$ the -a$( e %ata Pro(erties dialog set the $u#ber o data ro7s a$d !olu#$s9 Bou !a$ s%e!i y 7hether the ro7s or the !olu#$s re%rese$ts sa#%les2 a$d o%tio$ally s%e!i y the ty%e o data :e9g9 *ata ty%e5bu$da$!e;2 7hi!h si#%ly allo7s 3. to !hoose $atural de aults9 5dd #ea$i$g ul :a$d u$i8ue; sa#%le a$d 3ariable labels usi$g2 or eAa#%le Edit*.a)e s*-a$( es. 4a3e the sheet usi$g +i e*-ave %ata As a$d sa3e re8ue$tly as you edit9 ?he de ault ile ty%e is a bi$ary PRIMER . 7orksheet or#at :9%ri;2 7hi!h is $ot ba!k7ards !o#%atible :i9e9 !a$$ot be read by PRIMER 352 though 35 9%ri iles !a$ be read by 3.;9 Bou !a$ also sa3e the data i$ EA!el or#at9 4o#e a$alyses re8uire %rior ide$ti i!atio$ o the sa#%les i$to grou%s2 e9g9 di ere$t sites2 ti#es2 treat#e$ts et!9 ?his i$ or#atio$ is held 7ith the 7orksheet i$ the or# o o$e or #ore a!tors :e9g9 ti#e; 7ith di ere$t le3els :e9g9 Na$2 6eb2 Mar2 Q;9 Bou !a$ !reate or #odi y this i$ or#atio$ by the Edit*+actors #e$u2 7he$ the a!ti3e 7orksheet is the data #atriA9 4i#ilarly2 grou%i$g stru!tures o$ the 3ariables are !reated usi$g Edit*Indicators9 Note that you !a$ get the ite#s o$ the Edit #e$u by right-!li!ki$g 7ith the #ouse 7he$ the !ursor is o3er the 7orksheet9 +se Edit*+actors to look at the a!tor C*istD already set u% or the ek#a9%ri 7orksheet9 ?his grou%s the sa#%les i$to o$e o our !lasses2 de%e$di$g o$ ho7 !lose they are to the !e$tre o drilli$g a!ti3ity9 ?he$ re3ert ba!k to the data 7orksheet by Cance or O/9 :?he our le3els2 5 to *2 are de i$ed i$ the *es!ri%tio$ boA u$der Edit*Pro(erties;9 Pre-treat#e$t o data Pre-treat#e$t o the data :so#eti#es i$ #ore tha$ o$e 7ay; is usually desirable9 6or asse#blage data2 tra$s or#atio$s 7ill redu!e the do#i$a$t !o$tributio$ o abu$da$t s%e!ies to ,ray-Curtis si#ilarities9 ?hough $ot usually $eeded or !o$trolled :C8ua$titati3eD; sa#%li$g2 sta$dardisi$g o
/

Readi$g i$ $o$3. iles

5dd a!tors or i$di!ators

sa#%les to relati3e !o#%ositio$ :so sa#%le totals are all (00F; !a$ be a!hie3ed by Ana 'se*Pre0 treat$ent*-tandardise*:4ta$dardise4a#%les;&:,y?otal;9 ?ra$s or#atio$ o all 3alues :7hi!h should be a ter sta$dardisatio$2 i the latter is a%%ro%riate; is a!hie3ed by2 or eAa#%le2 Ana 'se* Pre0treat$ent*&rans!or$ 1overa 2E?ra$s or#atio$" 48uare root9 6or e$3iro$#e$tal-ty%e data2 su!h as !o$ta#i$a$t !o$!e$tratio$s2 it #ay be a%%ro%riate to tra$s or# to a%%roAi#ately $or#al distributio$s :to Lusti y the use o a Eu!lidea$ dista$!e #easure;2 a$d the$ to $or#alise :a lo!atio$ a$d s!ale !ha$ge; to %la!e di ere$t ty%es o 3ariables o$to a !o##o$ #easure#e$t s!ale9 4ee the Methods #a$ual or the ratio$ale or 3arious !hoi!es9 -ther %re-treat#e$t o%tio$s i$!lude di ere$tial 7eighti$g o ea!h 3ariable :3ia su%%lied 7eights or by !al!ulati$g dis%ersio$ %ro%erties o re%li!ates;9 Ea!h %re-treat#e$t o%eratio$ !reates a $e7 7orksheet o tra$s or#ed>sta$dardised> $or#alised data2 a$d so#eti#es the o%tio$ to se$d su##ary statisti!s to a $e7 7orksheet :e9g9 the #ea$ a$d sta$dard de3iatio$ or ea!h 3ariable2 7he$ taki$g Ana 'se*Pre0treat$ent*,or$a ise;9 &orki$g 7ith rese#bla$!es CRese#bla$!eD is the ge$eral ter# !o3eri$g si#ilarity2 dissi#ilarity or dista$!e !oe i!ie$ts9 Bou !a$ !reate a rese#bla$!e #atriA 7ith the Ana 'se*Rese$) ance #e$u ite#9 4i#ilarity #atri!es !a$ be sa3ed as i$di3idual PRIMER or#at iles :they 7ill be gi3e$ a 9sid eAte$sio$2 as i$ 35;2 but this is rarely $e!essary i$ 3.2 si$!e it is $o7 %ossible to sa3e the 7hole 7orks%a!e9 Cli!k o$ the title bar o the ek#a9%ri 7i$do72 o$e o the iles you o%e$ed earlier2 to #ake it the a!ti3e 7i$do7 K the title bar be!o#es a #ore %ro#i$e$t !olour or the a!ti3e 7i$do79 Pre-treat the data by a s8uare root tra$s or#atio$9 &ith the tra$s or#ed #atriA as the a!ti3e sheet2 sele!t Ana 'se*Rese$) ance a$d take the de aults o :5$alyse bet7ee$ 4a#%les; & :Measure,rayCurtis si#ilarity;9 ?his !reates a tria$gular #atriA o si#ilarities bet7ee$ e3ery %air o sa#%les2 i$ a $e7 7i$do72 7hi!h $o7 be!o#es the a!ti3e sheet or se3eral urther a$alyses9 :?here are a 7ide ra$ge o other rese#bla$!e !oe i!ie$ts that !a$ be a!!essed 3ia the More butto$ a$d tab9 ?he #easures that a%%ear i$ the ge$eral re ere$!e book o Lege$dre P & Lege$dre L2 (<<=2 Nu#eri!al e!ology2 2$d E$glish ed2 Else3ier2 are $u#bered i$ the sa#e 7ay as there9; Creati$g %lots &he$ a rese#bla$!e #atriA is the a!ti3e 7i$do72 the Ana 'se #e$u s7it!hes to dis%lay o$ly the a$alyses 7hi!h 7ork 7ith rese#bla$!es :e9g9 Ana 'se*C.3-&ER or *M%-;9 Ru$$i$g routi$es o$ the 5$alyse #e$u %rodu!es gra%hi!al out%ut2 results 7i$do7s a$d>or urther 7orksheets9 ?he %ro%erties o %lots !a$ be a#e$ded through the Gra(h #e$u9 Bou !a$ also get the gra%h #e$u ite#s by right !li!ki$g the #ouse 7he$ the !ursor is o3er the gra%hi!s 7i$do79 Plots !a$ be sa3ed i$ PRIMER or#at :9%%l; but #ore use ully i$ se3eral eA%ort or#s2 either %iAel-based :9b#%2 9L%g2 9%$g2 9ti ; or 3e!tor-based &i$do7s #eta iles :9e# ; or urther #a$u%ulatio$ i$ Po7er%oi$t et!9 Ge$erate a de$drogra# or a !luster a$alysis usi$g the ,ray-Curtis si#ilarity #atriA Lust %rodu!ed9 &ith this as the a!ti3e 7i$do72 take Ana 'se*C.3-&ER*MainECluster #odeGrou% a3erage2 a$d the Plot de$drogra# !he!k boA ti!ked :but $ot the 4IMPR-6 boA2 or $o7;9 Cli!k o$ the O/ butto$ to %rodu!e the %lot9 ?ry alteri$g the %lotDs a%%eara$!e9 Cli!k o$ a horiIo$tal li$e to rotate %arts o the de$drogra#2 as i it 7ere a C#obileD :de#o$strati$g the relati3e arbitrari$ess o the A aAis orderi$g;2 a$d !li!k o$ a 3erti!al li$e to !olla%se the tree belo7 that %oi$t9 Rei$state by !li!ki$g agai$9 5dd sy#bols to the A aAis2 re%rese$ti$g the our %re-de i$ed dista$!e grou%s or sa#%les ro# the oil ield2 by Gra(h*%ata a)e s 4 s'$)o s*:4y#bols"PlotE,y a!torE*ist ; & :Labels" Plot;9 EA%eri#e$t 7ith other o%tio$s2 e9g9 !ha$gi$g title siIes a$d o$ts o$ the &it es tab2 u$!he!ki$g the Plot history boA o$ the Genera tab2 -rie$tatio$Right o$ Gra(h*-(ecia et!9 EA%lori$g the 7orks%a!e ,y $o72 you ha3e !reated #a$y o%e$ 7i$do7s a$d #ay be i$di$g it hard to see the o$e o i$terest by shu li$g the 7i$do7s arou$d9 ?his is #ore easily do$e by !leari$g the PRIMER deskto% 7ith Window*C ose A Windows :this does $ot re#o3e the iles ro# the 7orks%a!e2 u$like the o%eratio$ o 35;2 a$d the$ usi$g the EA%lorer tree o$ the le t o the deskto% to ide$ti y the %lots2 results2 7orksheets et! that you 7a$t to 3ie7 :!li!k o$ their $a#e;9 Bou !a$ re$a#e a$y o these ite#s by +i e*Rena$e5 or2 7he$ the !ursor is o3er a $a#e o$ the EA%lorer tree the$ right !li!ki$g o$ the #ouse gi3es you -ave2 Rena$e or %e ete o%tio$s :the latter also re#o3es ro# the 7orks%a!e all ite#s that bra$!h do7$ ro# the deleted o$e;9 ?he bra$!hi$g stru!ture is $ot Lust or

dis%lay K PRIMER . uses it hea3ily to suggest se$sible de aults a$d to %ass i$ or#atio$2 su!h as $e7ly-!reated a!tors2 do7$ the tree to use i$ gra%hs2 or e3e$ u% the tree to the origi$al dataM &orks%a!es !a$ be sa3ed i$ their e$tirety9 *o this or the !urre$t 7orks%a!e 7ith +i e*-ave Wor6s(ace As*:6ile $a#e" ?est(;H 7orks%a!e iles ha3e eAte$sio$ 9%7k a$d a di ere$t i!o$ to data2 rese#bla$!e or gra%h iles9 No7 +i e*C ose Wor6s(ace the$ re-o%e$ it agai$ 7ith +i e* O(en*6ile $a#e" ?est(9%7k9 It re-a%%ears exactly as it 7as sa3ed :it has $o dy$a#i! li$ks to iles outside the 7orks%a!e2 so subse8ue$t deleti$g>editi$g o these 7ill $ot !ha$ge the 7orks%a!e data;9 4ele!ti$g data subsets 4o#eti#es you #ay 7ish to o#it !ertai$ s%e!ies or sele!t o$ly a subset o sa#%les or a$alysis9 Bou !a$ a%%ly sele!tio$ to o$e or both o sa#%les a$d 3ariables by irst highlighi$g the ro7s a$d> or !olu#$s2 by !li!ki$g :a$d draggi$g; o$ the ro7 or !olu#$ labels9 ?hese o%eratio$s are toggled2 so are !a$!elled by a si#%le re%eat9 :5 use ul tri!k is to !li!k i$ the bla$k boA i$ the to% le t !or$er o the 7orksheet2 7hi!h toggles bet7ee$ highlighti$g all a$d highlighti$g $o$e9; &ith the !orre!t subset highlighted :the darkest shaded areas;2 !hoose -e ect*7igh ighted9 ?he sele!ted subset is dis%layed a$d a$alysis o%erates o$ly o$ this subset9 Re3ert later to the ull 7orksheet by -e ect* A a$d you !a$ Edit*C ear 7igh ight :a$other use ul tri!k is Edit*Invert 7igh ight a$d -e ect* 7igh ighted agai$2 to get the !o#%le#e$tary set o 3ariables>sa#%les to the o$e you had origi$ally sele!ted;9 Note that i you use this #ethod o sele!tio$ you #ust re#e#ber that highlighting alone is not selection" you #ust irst highlight the$ sele!t the highlights9 5lter$ati3ely2 sele!t sa#%les :or 3ariables; by their $u#bers :e9g9 -e ect*-a$( es*4a#%le $u#bers" (-52((-(5; or by le3els o a a!tor :e9g9 -e ect*-a$( es*6a!tor le3elsE6a!tor $a#e" *istE.eve s*I$!lude" *;9 5$y sele!tio$ is o$ly te#%orary K the sele!ted %ortio$ is dis%layed but all the origi$al data a$d a!tors are still there2 i$ the ba!kgrou$d9 4a3i$g the !urre$t sheet to a ile 7ill sa3e the ull data :this is to sto% you o3er7riti$g your ull data 7ith a subset2 by #istake;9 ?o sa3e o$ly the sele!ted %ortio$ to a ile2 you #ust irst !o%y it to a $e7 sheet2 7ith &oo s*%#( icate9 -$ly the sele!ted %ortio$ is du%li!ated9 ?his is also the 7ay you !a$ do !rossed sele!tio$s usi$g a!tor le3els2 e9g9 sele!t a subset o the sites2 du%li!ate this sheet2 the$ sele!t a subset o ti#es o$ the du%li!ated sheet9 ?ools #e$u %#( icate is Lust o$e o se3eral routi$es to #a$i%ulate your data or rese#bla$!e 7orksheets %rior to :or bet7ee$; a$alyses2 usi$g the &oo s #e$u9 &he$ the a!ti3e #atriA is sa#%le data the other !hoi!es !o3er" Aggregate2 7hi!h uses a di ere$t or# o 7orksheet2 a$ aggregatio$ ile :de i$i$g 7hi!h s%e!ies belo$g to 7hi!h ge$era2 7hi!h a#ilies et!; to %ool s%e!ies !ou$ts2 or eAa#%le2 to higher taAo$o#i! le3elsH Average a$d -#$2 7hi!h are #ost !o##o$ly used to a3erage or total sa#%les 7ith the sa#e a!tor le3el :e9g9 a3erage asse#blages or ea!h site2 a!ross all ti#es;H Chec62 7hi!h ide$ti ies %ote$tially %roble#ati! data e$tries or so#e a$alysis !hoi!es :sa#%les 7hi!h are all Iero2 $o$-u$i8ue sa#%le $a#es2 #issi$g 3alues2 $egati3e 3alues et!;H Merge2 a !o#%rehe$si3e Loi$i$g tool2 7hi!h 7ill !o#bi$e t7o 7orksheets i$to o$e2 usi$g the sa#%le a$d 3ariable labels to ide$ti y ho7 the Loi$ is #ade :e9g9 t7o arrays o di ere$t sa#%les but o$ly %artially o3erla%%i$g s%e!ies sets !a$ be #erged i$to a u$i ied #atriA K !o$siste$t s%elli$g o labels is i#%orta$t hereM;H Missing2 7hi!h %ro3ides Cbest %redi!tio$sD o isolated #issi$g 3alues K #aAi#u# likelihood esti#ates ro# the EM algorith# K u$der stri!t #odel !o$ditio$s :use 7ith great !areM;H a$d Ran6 varia) es2 7hi!h !a$ be a use ul alter$ati3e2 or %la!i$g e$3iro$#e$tal-ty%e data o$ a !o##o$ #easure#e$t s!ale2 to $or#alisatio$ a ter &rans!or$ 1individ#a 2 o%eratio$s are !arried out9 ?his i$di3idual tra$s or# o%tio$ greatly eA%a$ds the li#ited ra$ge o o3erall tra$s or#s o$ the Ana 'se*Pre0treat$ent*&rans!or$ 1overa 2 #e$u9 ?he latter are a%%lied i$ the sa#e 7ay to all data i$ the sheet2 but the i$di3idual tra$s or#s a%%ly o$ly to all 3alues :J; i$ a highlighted set o 3ariables2 a$d the user #ay build a s%e!ialised tra$s or# eA%ressio$2 usi$g s!ie$ti i! u$!tio$s2 o%erators2 other 3ariables et!9 6i$ally2 &rans(ose s7it!hes ro7s a$d !olu#$s or the %ur%ose o eA%orti$g to a$other a%%li!atio$ :$ote" this is $ot the sa#e thi$g as !ha$gi$g the desig$atio$ o 7hi!h labels re%rese$t sa#%les a$d 7hi!h 3ariables2 3ia Edit*Pro(erties;9 ?he out%ut o &oo s are usually $e7 7orksheets2 7hi!h be!o#e the i$%ut to a$ Ana 'se o%tio$9 4o#e o the sa#e #e$u ite#s e9g9 %#( icate2 &rans!or$2 a%%ear u$der the &oo s #e$u 7he$ the a!ti3e sheet is a rese#bla$!e #atriA2 but others are s%e!i i! to rese#bla$!es2 e9g9 %issi$i arit' !o$3erts si#ilarities to dissi#ilarities :a$d 3i!e-3ersa;2 by subtra!ti$g ro# (009 ?his is really o$ly $eeded or eA%orti$g to other a%%li!atio$s si$!e i$ter$ally PRIMER k$o7s 7hi!h #atri!es are o
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7hi!h ty%e :si#ilarity2 dissi#ilarity or dista$!e;9 It 7ill auto#ati!ally #at!h the# i$ the right 7ay2 e9g9 i you try to !orrelate a biologi!al ,ray-Curtis si#ilarity #atriA to a$ e$3iro$#e$tal Eu!lidea$ dista$!e #atriA :usi$g Ana 'se*RE.A&E;2 lo7 dista$!es 7ill be #at!hed to high si#ilarities9 Results 7i$do7s Nearly all !hoi!es ro# the &oo s or Ana 'se #e$u 7ill %rodu!e teAt- or#at results9 ?hese are se%arate 7i$do7s2 de$oted by $ote%ad i!o$s i$ the EA%lorer tree9 It is thus easy to i$d a$d dis%lay the results ro# a$y %arti!ular a$alysis :the 35 si$gle large log o all results $o lo$ger eAists2 its %ur%ose bei$g #ore hel% ully take$ by the EA%lorer tree;9 I$di3idual results 7i$do7s !a$ be sa3ed to 9rt or 9tAt or#at iles but2 #ore si#%ly2 the usual &i$do7s Edit*Co(' :CtrlRC; 7orks o$ teAt i$ the results 7i$do7 that has bee$ highlighted2 e9g9 it !a$ easily be %asted to a &ord ile9 &he$ results tables are %asted i$to EA!el i$ this 7ay the e$tries are %la!ed i$to a grid auto#ati!ally9 I$ &ord iles2 they !a$ be !o$3erted ro# teAt to tables si#%ly by s%e!i yi$g tab as the se%arator9 &ith the ,ray-Curtis rese#bla$!e #atriA ro# the Eko isk data as the a!ti3e sheet2 ru$ the o$e7ay 5N-4IM test o$ the our grou%s o sa#%les at di ere$t dista$!es ro# the oil ield :the a!tor *ist;2 by Ana 'se*A,O-IM*Genera *:*esig$-$e 7ayE6a!tor 5" *ist; & :MaA %er#utatio$s" <<<; & :Pair7ise tests or 7orksheet; & :Plot histogra#;9 ?hree $e7 7i$do7s are !reated9 6irstly2 the results 7i$do7 gi3es the 5N-4IM R statisti! :see Methods #a$ual2 Cha%ter .; or testi$g 7hether asse#blage stru!ture 3aries a!ross the our dista$!e grou%s2 a$d sho7s that it does9 ?he gra%h dis%lays the histogra# o the $ull :%er#utatio$; distributio$ o R2 a$d sho7s 7hy the $ull hy%othesis o C$o di ere$!e i$ asse#blage stru!ture bet7ee$ grou%sD is de!isi3ely reLe!ted :the obser3ed R is 7ell outside the $ull distributio$;9 ?he results 7i$do7 the$ gi3es the %air7ise tests bet7ee$ %airs o grou%s9 ?he 3alues o 5N-4IM R or ea!h %air ha3e also bee$ se$t to a urther tria$gular 7orksheet :7here they !ould2 or eAa#%le2 be sa3ed to EA!el or e3e$2 i$ #ore !o#%li!ated !ases2 be used as i$%ut to a urther a$alysis;9 5$alyse #e$u Mu!h o the abo3e is %re%aratory to the !ore routi$es i$ PRIMER2 ou$d o$ the Ana 'se #e$u2 7hose o%eratio$ is !o3ered i$ detail i$ the ollo7i$g !ha%ters2 a$d its ratio$ale i$ the asso!iated Methods #a$ual9 5ll that !a$ be gi3e$ here is a brie list9 &he$ the a!ti3e 7i$do7 is a sa#%le data sheet2 the !hoi!es i$!lude" "E-&2 sele!ti$g 3ariable subsets i$ o$e #atriA 7hi!h best #at!h the #ulti3ariate %atter$ o sa#%les i$ a di ere$t #atriA2 e9g9 CeA%lai$i$gD biologi!al asse#blage stru!ture 7ith a subset o e$3iro$#e$tal 3ariablesH .I,/&REE2 a $o$-%ara#etri! #ulti3ariate or# o C!lassi i!atio$ a$d regressio$ treesD2 ta!kli$g the sa#e %roble# as ,E4? but i$ a #ore %ie!e#eal 7ay2 e9g9 tryi$g to ide$ti y 7hi!h i$di3idual abioti! 3ariables2 a$d 7hat ra$ges o their 3alues2 a%%ear to be res%o$sible or dis!ri#i$ati$g di ere$t asse#blage grou%i$gsH %IVER-E2 !al!ulati$g a 7ide ra$ge o sta$dard di3ersity #easures2 i$!ludi$g so#e $e7er o$es based o$ the !o$!e%t o taAo$o#i! or %hyloge$eti! breadth o a$ asse#blageH PCA2 ordi$atio$ by Pri$!i%al Co#%o$e$ts2 #ore use ul or %hysi!al>!he#i!al>e!otoAi!ologi!al a$d si#ilar abioti! data tha$ or s%e!ies abu$da$!esH -IMPER Csi#ilarity %er!e$tagesD2 ide$ti yi$g 3ariables :e9g9 s%e!ies; that %ri#arily a!!ou$t or ,ray-Curtis dissi#ilarities or Eu!lidea$ dista$!es obser3ed bet7ee$ grou%s o sa#%lesH -IMPRO+2 Csi#ilarity %ro ilesD2 7hi!h test or sig$i i!a$t e3ide$!e o stru!ture a#o$g sa#%les that ha3e $o %re-de i$ed grou%i$g :this !a$ be !o#bi$ed 7ith both .I,/&REE a$d C.3-&ER to Lusti y ide$ti i!atio$ a$d i$ter%retatio$ o !lusters;H %ra!ts$an P ot2 a #ulti%le s!atter %lot o %airs o 3ariables :o te$ e$3iro$#e$tal;H %o$inance P ot a$d %OM%I-2 %rodu!i$g a$d testi$g grou%s o do#i$a$!e !ur3es a$d 5,C %lots2 7hi!h are gra%hi!al or#s o di3ersity #easures o$ s%e!ies abu$da$!e :a$d bio#ass;2 as is a Geo$etric C ass P ot9 6i$ally2 a -(ecies0 Acc#$ P ot 7ill gra%h s%e!ies-a!!u#ulatio$ !ur3es2 sho7i$g ho7 the obser3ed $u#ber o s%e!ies i$!reases as $e7 sa#%les are added2 a$d also %lotti$g a 7ide ra$ge o s%e!ies ri!h$ess esti#ators :the asy#%tote o s%e!ies-area !ur3es2 assu#i$g C!losedD !o##u$ities;9 4o#e o these routi$es :e9g9 ,E4? or 5,C !ur3es; re8uire t7o or #ore i$%ut 7orksheets" o$e is al7ays ide$ti ied as the a!ti3e 7i$do7 be ore the routi$e is ru$ :e9g9 the e$3iro$#e$tal #atriA that 7ill be sele!ted ro# i$ ,E4?2 or the abu$da$!e #atriA or 5,C !ur3es; a$d the other is su%%lied to the routi$e 3ia a dialog boA :e9g9 the !o##u$ity si#ilarity #atriA i$ ,E4?2 or the bio#ass sheet i$ 5,C !ur3es;9 &he$ the a!ti3e 7i$do7 is a rese#bla$!e #atriA2 the Ana 'se #e$u !o3ers :as 7ell as C.3-&ER a$d A,O-IM already #et;" M%- ordi$atio$ %lots2 $o$-#etri! #ultidi#e$sio$al s!ali$g2 7hi!h is !e$tral to the PRIMER a%%roa!hH MV%I-P2 7hi!h !o#%ares #ulti3ariate dis%ersio$ a!ross grou%sH RE.A&E2 !o3eri$g $o$-%ara#etri! Ma$tel-ty%e tests bet7ee$ rese#bla$!e #atri!esH a$d 8-&AGE2 a se!o$d-stage a$alysis :a$ M*4 o M*4DsM;2 use ul or !o#%ari$g the e e!ts o
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di ere$t !hoi!es o data aggregatio$2 tra$s or#atio$2 rese#bla$!e et!9 -$e routi$e2 &A9%&E-&2 is a!!essed through the Ana 'se #e$u 7he$ a$ aggregatio$ #atriA is the a!ti3e 7i$do7" this tests biodi3ersity #easures based o$ taAo$o#i! disti$!t$ess or de%artures ro# CeA%e!tatio$D9

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