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Evidence for reproductive interference between sexual and apomictic populations of the Easter Daisy (Townsendia… Garani, Alice 2014

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Evidence(for(reproductive(interference(between(sexual(and(apomictic(populations(of(the(Easter(Daisy((Townsendia*hookeri,(Asteraceae)(by#Alice#Garani#B.Sc.#(Hons),#The#University#of#Bologna,#2009#A#THESIS#SUBMITTED#IN#PARTIAL#FULFILLMENT#OF#THE#REQUIREMENTS#FOR#THE#DEGREE#OF#MASTER#OF#SCIENCE#in#THE#FACULTY#OF#GRADUATE#AND#POSTDOCTORAL#STUDIES#(Botany)#THE#UNIVERSITY#OF#BRITISH#COLUMBIA#(Vancouver)#September#2014#©#Alice#Garani,#2014#ii Abstract(Townsendia#hookeri#(Asteraceae)#plants#can#reproduce#sexually#or#via#apomixis#(i.e.#asexual#reproduction#through#seeds),#and#the#breeding#system#is#tightly#linked#with#ploidy#level,#so#that#sexual#outcrossers#are#exclusively#diploid#while#apomictic#plants#are#polyploid.#The#species#grows#from#central#Colorado#to#British#Columbia,#with#a#disjunct#distribution#in#Yukon#Territory.#Outside#Yukon,#sexual#populations#are#restricted#to#the#southernmost#portion#of#the#range#(Colorado#and#southern#Wyoming)#and#apomictic#populations#occur#from#Wyoming#to#Canada,#a#pattern#consistent#with#geographical#parthenogenesis.#The#major#objective#of#this#study#was#to#expand#our#understanding#of#the#factors#that#have#shaped#and#maintain#this#distribution,#conferring#an#apparent#advantage#to#apomictic#lineages#over#their#sexual#progenitors.#Having#documented#that#polyploid#plants#retain#the#ability#to#produce#some#functional#pollen,#I#hypothesized#that#if#sexual#forms#spread#into#an#apomictic#population,#and#thus#receive#mostly#heterospecific#pollen,#they#would#have#reduced#reproductive#success,#because#the#progeny#sired#in#sexual[asexual#crosses#are#predicted#to#be#weak#or#inviable,#and#to#include#hybrid#apomicts.#This#asymmetric#reproductive#interference#could#help#explain#why#diploids#have#failed#to#spread#into#the#territory#dominated#by#apomicts.##To#test#this#hypothesis#I#performed#a#cross[pollination#experiment#in#the#field.#I#showed#that#diploid#and#polyploid#cytotypes#have#comparable#reproductive#success#(at#least,#in#the#area#where#the#experiment#was#conducted),#and#I#also#confirmed#that#diploids#are#outcrossers#while#polyploids#are#apomictic.#The#crossing#experiment#indicates#that,#despite#its#low#viability,#pollen#produced#by#apomicts#can#fertilize#and#negatively#affect#diploid#seed#parents.#As#predicted,#when#sexual#plants#received#heterospecific#rather#than#conspecific#pollen,#seed#set#and#germination#rate#were#reduced,#and#seedlings#had#a#lower#survival#rate.#Flow#cytometric#analysis#of#offspring#from#sexual[asexual#crosses#revealed#the#presence#of#putative#euploid#(diploid,#triploid#and#tetraploid)#as#well#as#aneuploid#cytotypes.#Based#on#ploidy#level#it#is#reasonable#to#expect#that#polyploid#hybrids#inherited#apomictic#genes#and#that#at#least#some#of#them#might#be#fully#apomictic.#iii Preface(The#research#question#and#methodological#design#for#this#thesis#were#developed#collaboratively#by#my#supervisor,#Dr.#Jeannette#Whitton,#and#me.#I#directed#and#carried#out#most#of#the#field#work#for#this#project;#however#some#plant#materials#were#collected#in#the#field#by#Adam#Wilkinson.#Whitton#and#Wilkinson#assisted#me#with#the#crossing#experiment#in#2012,#and#Jamie#Leathem#assisted#in#2013.#Chris#Lee#and#A.#Wilkinson#helped#with#seed#collection.#I#also#carried#out#the#experiments#in#the#growth#chamber#and#the#flow#cytometric#analysis.#The#pollen#study#was#conducted#in#collaboration#with#C.#Lee,#Erica#Li[Leger#and#Alberto#Ruiz[Larrea.#I#completed#the#data#analysis#and#the#writing#of#the#thesis.#Dr.#Jeannette#Whitton,#Dr.#Roy#Turkington#and#Dr.#Sally#Aitken#assisted#with#interpretation#of#the#results#and#provided#helpful#feedback.##iv Table(of(contents(Abstract(...................................................................................................................................................................(ii#Preface(....................................................................................................................................................................(iii#Table(of(contents(.................................................................................................................................................(iv#List(of(tables(..........................................................................................................................................................(vi#List(of(figures(.....................................................................................................................................................(viii#Acknowledgements(............................................................................................................................................(xi#Chapter(1:(Introduction(.....................................................................................................................................(1#1.1# The#Easter#Daisy#(Townsendia#hookeri)#............................................................................................................#1#1.2# Apomixis#.........................................................................................................................................................................#2#1.3# Assessment#of#breeding#system:#pollen#analysis#and#flow#cytometry#(FCM)#..................................#4#1.4# Geographical#parthenogenesis#..............................................................................................................................#6#1.5# Hypotheses#for#the#occurrence#of#sexual#and#apomictic#populations#in#Townsendia#hookeri#..#7#1.6# Study#objectives#..........................................................................................................................................................#9#Chapter(2:(Could(reproductive(interference(limit(the(range(expansion(of(sexual(populations(of(Townsendia*hookeri*into(the(territory(occupied(by(their(polyploid(descendants?(...............(11#2.1# Introduction#...............................................................................................................................................................#11#2.2# Methods#.......................................................................................................................................................................#12#2.2.1# Study#site#..........................................................................................................................................................#12#2.2.2# Experimental#design#and#field#work#....................................................................................................#13#2.2.3# Estimation#of#reproductive#success#.....................................................................................................#15#2.2.3.1# Seed#set#...................................................................................................................................................#16#2.2.3.2# Average#achene#mass#(mg)#............................................................................................................#16#2.2.3.3# Germination#rate#.................................................................................................................................#16#2.2.3.4# Survival#rate#per#transplanted#seedling#...................................................................................#17#2.2.3.5# Statistical#analysis#..............................................................................................................................#18## #v 2.2.4# Flow#cytometry#(FCM)#...............................................................................................................................#19#2.2.4.1# Plant#material#.......................................................................................................................................#19#2.2.4.2# Ploidy[level#determination#.............................................................................................................#20#2.3# Results#..........................................................................................................................................................................#23#2.3.1# Reproductive#success#of#sexual#versus#apomictic#populations#under#natural#conditions#(C#and#I#treatments)#..................................................................................................................................#23#2.3.2# Experimental#crossings#(SS#and#SA#treatments)#............................................................................#24#2.3.3# Flow#cytometry#(FCM)#...............................................................................................................................#25#2.4# Discussion#...................................................................................................................................................................#27#2.4.1# Reproductive#success#of#sexual#versus#apomictic#populations#under#natural#conditions#(C#and#I#treatments)#..................................................................................................................................#28#2.4.2# Experimental#crossings#(SS#and#SA#treatments)#............................................................................#30#2.4.3# Flow#cytometry#(FCM)#...............................................................................................................................#32#Chapter(3:(Conclusions(....................................................................................................................................(56#3.1# General#conclusions#................................................................................................................................................#56#3.2# Implications#................................................................................................................................................................#57#3.3# Future#directions#.....................................................................................................................................................#59#References(...........................................................................................................................................................(61#Appendices(..........................................................................................................................................................(67#Appendix#A:#Range#wide#survey#of#T..hookeri#populations#using#pollen#analysis#...................................#67#A.1# Methods#...............................................................................................................................................................#67#A.2# Results#..................................................................................................................................................................#69#Appendix#B:#Range#wide#survey#of#T..hookeri#populations#using#flow#cytometry#..................................#70#B.1# Methods#...............................................................................................................................................................#70#B.2# Results#..................................................................................................................................................................#71#Appendix#C:#Results#from#the#co[ground#test#(FCM)#............................................................................................#72# vi List(of(tables(Table(2.1#Identification#numbers,#site#descriptions#(with#GPS#coordinates#and#altitude),#pollen#data#(stainability,#average#size#and#standard#deviation#of#viable#grains)#and#ploidy#estimates#for#the#populations#of#Townsendia.hookeri#selected#to#conduct#the#cross[pollination#experiment,#including#population#2031#(described#in#chapter#3).#Pollen#stainability#and#size#were#measured#following#the#methodology#described#in#Appendix#A.#........................................................................................................................#38#Table(2.2#Summary#of#the#work#completed#in#the#field#during#the#spring#of#2012#and#2013.#For#each#population,#the#table#reports:#the#identification#number#(ID),#function#(SR#=#sexual#recipient,#SD#=#sexual#donor,#AD#=#apomictic#donor),#replicate#experiment#(R1#or#R2),#treatment(s)#done,#year#when#the#treatment(s)#was#conducted#and#number#of#days#over#which#the#pollination#was#repeated.#.........................................................................................................................................................................................................#40#Table(2.3#Summary#of#two[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#treatment#(control#vs.#isolation),#reproduction#(sexual#vs.#apomictic#breeding#system)#and#the#interaction#term#on#seed#set.#The#analysis#was#done#using#GLM#with#family#=#quasibinomial#to#account#for#overdispersion#(meaning#that#the#variance#of#the#error#distribution#in#the#data#was#greater#than#that#expected#under#the#binomial#distribution).#The#significance#of#terms#was#assessed#with#an#F[test.(Bold#values#are#significant#at#p<0.05.#..................................................................................................................#40#Table(2.4#Summary#of#one[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#reproduction#(sexual#vs.#apomictic#breeding#system)#on#germination#rate.#The#analysis#was#done#using#GLM#with#family#=#quasibinomial.#The#significance#of#terms#was#assessed#with#an#F[test.#Bold#value#is#significant#at#p<0.05.#.............................................................................................................................................................#41#Table(2.5#Summary#of#one[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#reproduction#(sexual#vs.#apomictic#breeding#system)#on#seedling#survival#rate.#The#analysis#was#done#using#GLM#with#family#=#binomial.#The#significance#of#terms#was#assessed#with#a#Chi[square#test.#Bold#value#is#significant#at#p<0.05.#.............................................................................................................................................................#41#Table(2.6#Summary#of#one[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#treatment#(sexual[sexual#crosses#vs.#sexual[apomictic#crosses)#on#seed#set#for#the#two#pollen#recipient#populations.#The#analysis#was#done#using#GLM#with#family#=#quasibinomial.#The#significance#of#terms#was#assessed#with#an#F[test.#Bold#value#is#significant#at#p<0.05.#........................................................#41#Table(2.7#Summary#of#one[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#treatment#(sexual[sexual#crosses#vs.#sexual[apomictic#crosses)#on#germination#rate#for#the#two#pollen#recipient#populations.#The#analysis#was#done#using#GLM#with#family#=#binomial.#The#significance#of#terms#was#assessed#with#a#Chi[square#test.#Bold#value#is#significant#at#p<0.05.#........................................#42#vii Table(2.8#Summary#of#one[way#factorial#Analysis#of#Deviance#testing#for#the#effects#of#treatment#(sexual[sexual#crosses#vs.#sexual[apomictic#crosses)#on#seedling#survival#rate#for#the#two#pollen#recipient#populations.#The#analysis#was#done#using#GLM#with#family#=#binomial.#The#significance#of#terms#was#assessed#with#a#Chi[square#test.#Bold#values#are#significant#at#p<0.05.#..................................#42#!viii List(of(figures(Figure(1.1#Flowering#plant#of#Townsendia.hookeri.#The#picture#was#taken#in#the#Medicine#Bow#National#Forest#(Wyoming,#U.S.)#in#April#of#2012.#...................................................................................................#10#Figure(1.2#Plant#of#Townsendia.hookeri#at#fruiting#stage,#showing#a#typical#seed#head.#The#picture#was#taken#in#the#Medicine#Bow#National#Forest#(Wyoming,#U.S.)#in#June#of#2012.#...................................#10#Figure(2.1#Locations#of#the#six#populations#of#Townsendia.hookeri#used#to#complete#the#cross[pollination#experiment.#Apomictic#populations#(IDs:#2019#and#2027)#are#represented#with#blue#circles#and#sexual#populations#(IDs:#2030,#2032,#2033#and#2035)#are#represented#with#red#circles.#The#figure#also#shows#the#location#of#the#apomictic#population#2031#(described#in#section#2.2.2).#.#43#Figure(2.2#One#replicate#block#of#experimental#treatments#completed#in#the#field#on#three#natural#populations#of#Townsendia.hookeri.#Treatments#are#indicated#with#capital#letters:#C,#control;#I,#isolation;#SS,#sexual[sexual#cross;#SA,#sexual[apomictic#cross.#The#number#of#individuals#exposed#to#each#treatment#is#indicated#in#parenthesis.#Plants#are#represented#as#having#a#single#inflorescence#to#indicate#that#only#one#flower#head#per#individual#was#used#in#a#given#treatment.#Blue#inflorescences#represent#apomictic#plants#while#red#inflorescences#represent#sexual#plants.#............#44#Figure(2.3(Achenes#of#Townsendia.hookeri#were#classified#as#“filled”#or#“empty”#based#on#the#following#criteria:#“filled”#achenes#were#plump#and#dark#brown#(first#and#second#achenes#from#the#left),#whereas#“empty”#achenes#were#flat#and#lacked#pigmentation#(fifth#and#sixth#achenes#from#the#left).#Light#brown#achenes#were#scored#as#“filled”#when#fleshy#(third#achene#from#the#left)#and#as#“empty”#when#clearly#devoid#of#an#embryo#(fourth#achene#from#the#left).#...................................................#45#Figure(2.4#Example#of#fully#established#individual#of#Townsendia.hookeri,#characterized#by#a#large#root#system#and#five#true#leaves.#.....................................................................................................................................#46#Figure(2.5#Cytogram#of#the#transit#time#of#propidium#iodide[stained#nuclei#through#the#laser#interrogation#point#(FL2[W)#versus#their#total#fluorescence#(FL2[A),#obtained#via#flow#cytometry.#This#type#of#plot#was#created#using#FlowJo#vX.0.6#and#it#was#used#to#identify#G0/G1#populations#of#nuclei#belonging#to#the#internal#standard#(Vicia.faba.cv.#Inovec#in#this#example)#and#the#target#tissue#(polyploid#Townsendia.hookeri).#The#two#subpopulations#of#nuclei#(in#light#green)#were#isolated#from#the#debris#background#(in#blue)#using#the#Autogate#function.#..............................................#47#Figure(2.6#Total#number#of#filled#and#empty#achenes#per#seed#head#(capitulum).#Each#head#(indicated#by#a#circle)#represents#an#apomictic#(blue)#or#sexual#(red)#seed#parental#plant#under#control#or#isolation#treatment.#The#black#circles#and#bars#are#means#and#standard#errors.#Different#shades#of#color#define#single#populations:#2019#is#dark#blue,#2027#and#2031#are#light#blue,#2030#is#dark#red#and#2033#is#light#red.#..........................................................................................................................................#48#ix Figure(2.7#Percentage#of#filled#achenes#per#seed#head#(seed#set).#Each#head#(indicated#by#a#circle)#represents#an#apomictic#(blue)#or#sexual#(red)#seed#parental#plant#under#control#or#isolation#treatment.#The#black#circles#and#bars#are#means#and#standard#errors.#The#asterisk#(*)#represents#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance.#Different#shades#of#color#define#single#populations:#2019#is#dark#blue,#2027#and#2031#are#light#blue,#2030#is#dark#red#and#2033#is#light#red.#......................................................................................................................................................................#49#Figure(2.8#Percentage#germination#rate#per#filled#achene.#Each#circle#represents#the#mean#value#for#an#apomictic#(blue)#or#sexual#(red)#seed#parental#plant#under#control#treatment.#The#black#circles#and#bars#are#means#and#standard#errors.#The#asterisk#(*)#represents#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance.#Different#shades#of#color#define#single#populations:#2019#is#dark#blue,#2027#is#light#blue,#2030#is#dark#red#and#2033#is#light#red.#.............................................#50#Figure(2.9#Percentage#survival#rate#per#seedling#transplanted#from#agar#to#soil.#Each#circle#represents#the#mean#value#for#an#apomictic#(blue)#or#sexual#(red)#seed#parental#plant#under#control#treatment.#The#black#circles#and#bars#are#means#and#standard#errors.#The#asterisk#(*)#represents#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance.#Different#shades#of#color#define#single#populations:#2019#is#dark#blue,#2027#is#light#blue,#2030#is#dark#red#and#2033#is#light#red.#.................................................................................................................................................................................................#50#Figure(2.10#Percentage#of#filled#achenes#per#seed#head#(seed#set).#Each#head#(indicated#by#a#circle)#represents#a#seed#parental#plant#under#sexual[sexual#(SS)#or#sexual[apomictic#(SA)#crossing#treatment.#Dark#grey#circles#correspond#to#population#2030,#while#light#grey#circles#correspond#to#population#2033.#The#red#circles#and#bars#are#means#and#standard#errors.#The#asterisk#(*)#represents#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance#done#between#treatments#within#each#population.#................................................................................................................................#51#Figure(2.11#Average#mass#(in#mg)#of#filled#achenes#per#seed#head.#Each#head#(indicated#by#a#circle)#represents#a#seed#parental#plant#under#sexual[sexual#(SS)#or#sexual[apomictic#(SA)#crossing#treatment.#Dark#grey#circles#correspond#to#population#2030,#while#light#grey#circles#correspond#to#population#2033.#The#red#circles#and#bars#are#means#and#standard#errors.#Differences#between#treatments#within#each#population#are#not#significant#based#on#the#T[test#(2030:#t=[0.3347,#df=46,#P=0.7393;#2033:#t=1.7632,#df=37,#P=0.0861).#...........................................................................................................#52#Figure(2.12#Percentage#germination#rate#per#filled#achene.#Each#circle#represents#the#mean#value#for#a#seed#parental#plant#under#sexual[sexual#(SS)#or#sexual[apomictic#(SA)#crossing#treatment.#Dark#grey#circles#correspond#to#population#2030,#while#light#grey#circles#correspond#to#population#2033.#The#red#circles#and#bars#are#means#and#standard#errors.#The#asterisk#(*)#represents#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance#done#between#treatments#within#each#population.#........................................................................................................................................................#53#x Figure(2.13#Percentage#survival#rate#per#seedling#transplanted#from#agar#to#soil.#Each#circle#represents#the#mean#value#for#a#seed#parental#plant#under#sexual[sexual#(SS)#or#sexual[apomictic#(SA)#crossing#treatment.#Dark#grey#circles#correspond#to#population#2030,#while#light#grey#circles#correspond#to#population#2033.#The#red#circles#and#bars#are#means#and#standard#errors.#The#asterisks#(*)#represent#significantly#different#values#(p<0.05)#based#on#the#Analysis#of#Deviance#done#between#treatments#within#each#population.#.................................................................................................#54#Figure(2.14#Genome#size#(2C[value)#of#plants#from#seed#or#pollen#parental#populations#(including#2031),#calculated#performing#flow#cytometry#on#silica[dried#leaf#tissue.#The#number#of#samples#analyzed#for#each#population#(n)#is#reported#in#the#legend.#Dotted#lines#delimit#size#ranges#associated#with#diploid#(2C[v.#<#14#pg;#in#red)#and#triploid#(17#pg#<#2C[v.#<#21#pg;#in#blue)#genomes.#Data#points#are#spread#along#the#y[axis#to#better#visualize#overlapping#values.#........................................#55#Figure(2.15#Genome#size#(2C[value)#of#offspring#from#sexual[apomictic#crosses#in#populations#2030#(n=41)#and#2033#(n=37),#calculated#performing#flow#cytometry#on#silica[dried#leaf#tissue.#Dotted#lines#delimit#size#ranges#associated#with#diploid#(11#pg#<#2C[v.#<#14#pg;#in#red)#and#polyploid#(2C[v.#>#17#pg;#in#blue)#genomes.#Orange#and#brown#circles#represent#nuclear#DNA#contents#consistent#with#an#aneuploid#condition.#Data#points#are#spread#along#the#y[axis#to#better#visualize#overlapping#values.#................................................................................................................................................................#55#Figure(A.2.1#Plot#of#mean#cytoplasm#diameter#of#viable#grains#by#mean#pollen#stainability,#representing#diploid#(dark#grey#circles)#and#polyploid#(white#circles)#cytotypes.#For#each#ploidy#category,#mean#stainability#(±standard#error)#and#mean#viable#pollen#size#(±standard#error)#were#found#to#be#equal#to:#82.81±2.63#%#and#22.31±0.25#µm#for#diploids#(n=24);#25.73±2.3#%#and#34.75±0.86#µm#for#polyploids#(n=34).#...........................................................................................................................#69#Figure(B.2.1#Genome#size#(2C[value)#of#68#samples#representing#25#populations#of#the#Easter#Daisy#(samples#from#the#same#population#always#cluster#together).#The#values#were#estimated#performing#flow#cytometry#on#silica[dried#leaf#tissue.#Samples#were#assigned#to#three#different#ploidy#levels:#diploid#(2Cx;#red#circles),#triploid#(3Cx;#blue#circles)#and#tetraploid#(4Cx;#light#blue#circles).#For#each#ploidy#category,#mean#2C[value#(±standard#deviation)#was#found#to#be#equal#to:#13.02±0.5#pg#for#diploids#(n=23),#18.34±0.89#pg#for#triploids#(n=33)#and#22.58±1.28#pg#for#tetraploids#(n=12).#.................................................................................................................................................................#71#!xi Acknowledgements(Foremost,#my#deepest#gratitude#is#to#Dr.#Jeannette#Whitton,#for#inspiring#and#facilitating#this#entire#thesis,#and#for#being#an#absolutely#fantastic#supervisor.#I#feel#very#privileged#to#have#had#the#opportunity#to#learn#from#her#and#the#other#excellent#researchers#of#the#UBC#Biodiversity#Research#Centre#and#the#Botany#Department.#Thank#you#to#Dr.#Roy#Turkington#and#Dr.#Sally#Aitken#who,#as#members#of#my#supervisory#committee,#offered#precious#advice#and#supported#me#throughout#my#graduate#work.#Dr.#Amy#Angert#helped#to#improve#the#clarity#of#the#dissertation#and#her#assistance#is#also#greatly#appreciated.#I#could#not#possibly#have#completed#all#the#experiments#I#did#in#the#field#without#the#assistance#of#my#lab#mates#and#brilliant#botanists:#Chris#Lee#and#Jamie#Leathem.#The#help#of#my#undergraduate#research#assistants#in#the#lab,#Erica#Li[Leger#and#Alberto#Ruiz[Larrea,#was#also#essential#to#this#project.#Dr.#Chris#Sears,#Dan#Bock#and#Andy#Johnson#were#of#invaluable#technical#help#during#the#development#of#the#flow#cytometry#protocol.#I#also#give#thanks#to#the#curators#of#the#UBC#Herbarium,#especially#Linda#Jennings#and#Amber#Saundry.#I#was#fortunate#to#have#the#support#and#input#of#a#number#of#talented#and#patient#colleagues#and#friends.#I#owe#particular#thanks#to#Dr.#Darren#Irwin,#Bill#Harrower,#Jenny#McCune#and#Sarah#Amundrud#for#their#advice#on#data#analyses.#Thank#you#also#to#Veronica#Oxtoby,#the#Botany#Department#graduate#student#secretary,#who#has#tirelessly#answered#my#questions#and#has#been#unwaveringly#helpful#for#many#years.##Special#thanks#to#my#friends#Daniela#Hegebarth,#Lisa#Boonzaier#and#Sara#Cocucci#who#have#guided#me#through#graduate#school#and#have#been#the#best#companions#I#could#have#asked#for.#Finally,#I#want#to#thank#my#family,#especially#Nedi#(la#mia#mamma)#for#her#enduring#and#unconditional#support#throughout,#and#Rafael#(my#adored#husband)#for#giving#me#his#love#and#encouragement,#and#for#making#many#personal#sacrifices#so#that#I#could#achieve#my#goals.# 1"Chapter(1: Introduction(The#establishment,#expansion#and#persistence#of#a#population#depend#in#part#on#the#ability#of#its#members#to#respond#to#spatial#and#temporal#environmental#variation.#Understanding#how#individuals#respond#to#such#variation#is#especially#challenging#for#taxa#that#include#both#apomictic#(i.e.,#clonal)#and#outcrossing#sexual#plants#(a#so#called#‘polyploid#agamic#complex’;#Babcock#and#Stebbins,#1938;#Grant,#1981),#because#it#is#unlikely#that#the#factors#that#promote#clonality#versus#outcrossing#would#diverge#among#closely#related#forms#over#short#distances.##The#complexity#of#these#systems#has#intrigued#researchers#for#many#decades#as#reflected#in#the#ongoing#debate#on#their#taxonomic#treatment#(e.g.,#Dickinson,#1998;#Hörandl,#1998)#and#in#our#poor#understanding#of#the#factors#that#determine#the#appearance#and#proliferation#of#most#agamic#complexes#(e.g.,#Hörandl#and#Hojsgaard,#2012).##Townsendia.hookeri#(the#Easter#Daisy)#is#a#well[documented#North#American#agamic#complex#(Beaman,#1957;#Thompson#and#Whitton,#2006)#that#provides#a#model#system#for#studying#the#origins#and#spread#of#apomixis.#An#experimental#investigation#of#reproductive#interactions#between#extant#sexual#and#apomictic#populations#of#the#Easter#Daisy#could#shed#light#on#the#circumstances#that#allow#the#persistence#of#sexual#individuals#and#successful#parthenogenetic#counterparts#in#nature.##1.1 The(Easter(Daisy((Townsendia(hookeri)(Named#by#William#Jackson#Hooker#in#honour#of#his#friend,#the#American#botanist#David#Townsend#(Larsen,#1927),#the#genus#Townsendia#(Asteraceae)#includes#approximately#thirty#annual,#biennial#or#perennial#herbs#native#to#western#North#America.#The#species#of#Townsendia#tend#to#have#limited#distributions,#to#the#point#that#“narrow#endemism#may#be#regarded#as#a#common#feature#of#2"the#genus”#(Beaman,#1957).#Townsendia#hookeri#occupies#a#relatively#wide#range,#occurring#from#the#base#of#the#Front#Range,#in#Colorado,#to#Yukon#Territory#in#northwestern#Canada.#The#species#is#also#known#as#Townsend’s#Easter#Daisy,#because#its#flowering#time#is#in#early#spring.#Townsendia#hookeri#is#a#diminutive,#densely#rosulate,#perennial#species#characterized#by#short#stems#that#end#with#tufts#of#narrow,#linear#leaves#and#sessile,#showy#inflorescences#(also#called#flower#heads#or#capitula)#(Figure#1.1).#Ray#florets#are#pistillate#with#white#to#pinkish#corollas,#whereas#disk#florets#are#hermaphroditic#with#yellow#corollas.#Pollen#is#released#when#the#style#elongates#pushing#out#through#the#anthers.#Fruits#are#achenes#(single[seeded,#indehiscent,#oblanceolate,#compressed#and#pubescent)#with#a#large#pappus#that#facilitates#wind[dispersal#(Beaman,#1957).#The#achenes#of#a#mature#inflorescence#form#a#round#cluster,#referred#to#as#‘seed#head’#(Figure#1.2),#and#tend#to#tumble#away#from#the#mother#plant#as#a#unit#until#they#gradually#detach#and#disperse#individually#(personal#observation).#Townsendia#hookeri#populations#typically#grow#about#2,000#m#above#sea#level#in#the#southernmost#portion#of#the#range,#and#their#elevation#declines#with#latitude#to#a#minimum#of#approximately#600#m#a.#s.#l.#in#Yukon#(Garani#et.al.,#unpublished#data).#The#species#thrives#on#xeric,#nutrient#poor,#sandy#soils,#along#road#cuts,#trails,#and#in#areas#of#natural#disturbance#(e.g.,#steep#hill#slopes)#with#sparse#and#usually#low#vegetation#cover,#and#patches#of#bare#soil.#1.2 Apomixis(In#roughly#half#of#the#taxa#within#Townsendia#individuals#can#reproduce#either#sexually#or#asexually#through#apomixis#(Beaman,#1957).#Here#used#as#a#synonym#of#agamospermy,(apomixis#describes#a#suite#of#processes#whereby#seeds#are#produced#from#unfertilized#ovules#(Richards,#1997).#When#the#development#of#the#embryo#presupposes#the#formation#of#an#unreduced#megagametophyte,#the#process#is#termed#gametophytic#apomixis#(Stebbins,#1950;#Nogler,#1984).#In#turn,#gametophytic#3"apomixis#can#be#further#subdivided#into#diplospory#and#apospory,#depending#on#the#cell#from#which#the#embryo#arises.#In#diplosporous#plants,#the#unreduced#embryo#sac#originates#from#the#megasporocyte,#which#undergoes#mitotic#or#mitotic[like#divisions,#instead#of#a#normal#reductional#meiosis.#In#aposporous#plants#the#unreduced#megagametophyte#arises#by#somatic#divisions#of#a#cell#of#the#nucellus#(Nogler,#1984;#Asker#and#Jerling,#1992;#Bicknell#and#Koltunow,#2004;#Whitton#et.al.,#2008).#In#both#cases,#the#unfertilized#and#unreduced#egg#cell#develops#into#an#embryo#through#parthenogenesis;#thus,#the#resulting#seed#generally#has#the#same#ploidy#level#and#genetic#content#as#the#maternal#parent.#Based#on#endosperm#initiation,#it#is#also#possible#to#distinguish#between#“autonomous”#and#“pseudogamous”#apomicts.#In#autonomous#apomixis,#endosperm#formation#is#independent#of#pollination,#while#in#pseudogamous#forms#the#fertilization#of#the#polar#nuclei#by#a#pollen#nucleus#is#essential#for#the#formation#of#viable#progeny#(Nogler,#1984;#Asker#and#Jerling,#1992;#Bicknell#and#Koltunow,#2004;#Whitton#et.al.,#2008).#Autonomous#endosperm#development#is#common#only#in#the#Asteraceae#(Nogler,#1984;#Mogie,#1992;#Noyes,#2007).##Through#his#embryological#studies#on#Townsendia,#Beaman#discovered#that#diplospory#prevails#in#the#genus,#and#that#embryo#and#seed#development#are#independent#of#pollination#(Beaman,#1957).#For#T..hookeri#in#particular,#apomixis#is#described#as#obligate#and#diplosporous,#with#autonomous#endosperm#formation.#Gametophytic#apomixis#in#plants#is#commonly#linked#with#polyploidy#(Asker#and#Jerling,#1992;#Whitton#et.al.,#2008),#and#available#evidence#indicates#that#this#is#so#in#T..hookeri.(Beaman,#1957;#Thompson#and#Whitton,#2006).#This#means#that#apomictic#individuals#are#exclusively#autopolyploid,#whereas#their#sexual#conspecifics#are#strict#outcrossing#diploids,#with#a#basic#chromosome#number#of#9#(thus,#2n=2x=18)#(Beaman,#1954;#Beaman,#1957).##Despite#being#obligate#and#autonomous#apomicts,#polyploid#individuals#of#the#Easter#Daisy#show#no#apparent#reduction#in#inflorescence#or#flower#size#compared#to#their#sexual#progenitors,#and#the#male#function#is#generally#maintained,#as#observed#in#most#plants#showing#apospory#or#diplospory#4"(Maynard#Smith,#1978;#Nogler,#1984).#Apomixis#only#affects#the#formation#and#development#of#the#female#gametophyte,#without#any#direct#influence#on#the#male#sporogenesis.#Nonetheless,#low#quality#pollen#is#often#produced,#especially#in#autonomous#apomicts,#where#disturbed#and#irregular#microsporogenesis#can#generate#highly#unbalanced#pollen#nuclei#and#sometime#leads#to#male#sterility#(Nogler,#1984;#Asker#and#Jerling,#1992).#Disruptive#effects#of#polyploidy#on#male#meiosis#are#likely#to#be#a#major#cause#of#this#failure.#Beaman#(1954;#1957)#observed#that#male#meiosis#is#normal#in#diploid#members#of#the#genus,#where#nine#bivalents#are#found#at#metaphase#I.#In#contrast,#meiotic#irregularities#are#frequent#in#polyploid#apomictics,#where#both#bivalents#and#univalent#are#found#at#metaphase#I,#and#“giant”#multi[nucleate#pollen#grains#are#occasionally#formed,#together#with#a#large#number#of#aborted#grains#of#different#sizes#(Beaman,#1954;#Beaman,#1957).#Thus,#as#observed#in#most#apomicts#with#only#rare#exceptions#(for#instance,#Meirmans#et.al.,#2006),#most#polyploid#members#of#T..hookeri#retain#the#ability#to#produce#some#functional#pollen.##1.3 Assessment(of(breeding(system:(pollen(analysis(and(flow(cytometry((FCM)(The#existence#of#clear#cytological#differences#between#the#pollen#of#sexual#and#apomictic#plants#has#been#exploited#to#discriminate#between#diploid#and#polyploid#cytotypes#of#the#Easter#Daisy.#Indeed,#with#the#exception#of#pollen#features,#sexual#and#apomictic#plants#do#not#possess#any#distinctive#morphological#character.#Pollen#size#and#stainability#(an#indicator#of#viability)#can#be#used#to#attribute#individual#samples#to#a#specific#breeding#type#(Beaman,1957;#Thompson#and#Whitton,#2006).#Using#these#methods,#we#have#conducted#a#range#wide#survey#of#populations,#examining#58#pollen#samples#obtained#from#herbarium#and#personal#collections,#including#desiccated#and#fresh#material#(Garani#et.al.,#unpublished;#Appendix#A).#In#accordance#with#previous#results#from#Thompson#and#Whitton#(2006),#this#survey#revealed#a#striking#contrast#between#the#small#(mean#diameter#and#standard#error:#22.31#±#0.25#µm)#and#highly#viable#(82.81#±#2.63#%#stainability)#5"pollen#typical#of#diploid#populations,#and#the#large,#irregular#(34.75#±#0.86#µm)#and#mainly#inviable#(25.73#±#2.3#%)#pollen#of#apomictic#populations#(Appendix#A).##Given#the#tight#association#between#ploidy#and#breeding#system#that#characterizes#T..hookeri,#it#is#also#possible#to#determine#the#mating#strategy#of#a#plant#by#measuring#its#genome#size.#Doležel#and#Bartoš#(2005)#showed#that#flow#cytometry#(FCM)#is#a#powerful#and#reliable#method#to#estimate#the#nuclear#genome#size#of#plants.#This#technique#indirectly#assesses#the#nuclear#DNA#content#of#a#tissue#by#measuring#the#degree#of#fluorescence#emitted#by#a#suspension#of#nuclei#stained#with#a#DNA[specific#fluorochrome.#This#approach#is#based#on#the#assumption#that#the#amount#of#light#emitted#by#the#stained#nuclei#has#a#linear#relationship#with#the#genome#size#(Doležel#and#Bartoš,#2005;#Doležel#et.al.,#2007).#The#nuclear#DNA#content#of#non[replicated#somatic#genome#measured#with#FCM#is#generally#termed#2C[value,#following#Greilhuber#et.al..(2005).#FCM#is#more#practical#than#chromosome#counting#because#it#can#be#applied#to#desiccated#leaf#tissue#(Suda#and#Trávníček#2006;#Sears,#2011),#sample#preparation#is#significantly#easier#and#a#large#number#of#nuclei#can#be#measured#in#a#short#time#(Sears,#2011).#The#range#wide#survey#carried#out#by#Garani#et.al.#(unpublished)#also#included#flow#cytometrical#analysis#of#68#samples#collected#from#25#populations#across#the#range,#excluding#Yukon#(described#in#Appendix#B).#Flow#cytometry#data#matched#the#expected#ploidy#levels#estimated#via#pollen#analysis,#but#also#revealed#that#nuclear#DNA#contents#of#polyploids#form#two#distinct#clusters,#suggesting#that#apomicts#can#be#either#triploid#or#tetraploid#(Appendix#B).#Combined#with#pollen#studies,#FCM#of#leaf#material#proved#to#be#a#powerful#tool#in#our#investigation#of#the#geographical#distribution#of#sexual#and#apomictic#populations#of#the#Easter#Daisy.## #6"1.4 Geographical(parthenogenesis(According#to#Beaman#(1957),#apomixis#must#represent#an#adaptive#advantage#in#some#of#the#species#of#Townsendia,#because#the#range#of#sexual#individuals#is#often#significantly#less#extensive#than#that#of#their#apomictic#counterparts.#This#is#especially#true#for#the#Easter#Daisy,#where#sexual#diploid#populations#are#restricted#to#the#southern,#historically#unglaciated#areas#of#the#range,#namely#Colorado#and#Wyoming,#with#a#few#sexual#populations#also#found#at#the#northern#limit,#in#Yukon.#In#contrast,#apomictic#populations#seem#to#be#excluded#from#the#territory#occupied#by#sexual#diploids,#but#have#established#further#north,#and#have#colonized#formerly#glaciated#portions#of#the#species’#range#(Beaman,#1957;#Thompson#and#Whitton,#2006;#Thompson#et.al.,#2008).#Moreover,#available#evidence#suggests#that#sexual#and#asexual#forms#do#not#occur#in#the#same#population.#The#geographical#distribution#of#sexual#and#apomictic#populations#of#T..hookeri#(excluding#Yukon)#has#been#recently#reassessed#using#pollen#cytology#and#FCM#(Garani#et.al.,#unpublished;#Appendices#A#and#B).#The#outcome#of#this#work#was#consistent#with#previous#findings,#but#it#also#showed#that#triploid#populations#occur#east#of#the#Rockies,#while#tetraploids#thrive#only#west#of#the#Great#Continental#Divide.#The#non[overlapping#distribution#of#sexuals#and#apomicts#is#consistent#with#a#pattern#known#as#geographical#parthenogenesis,#wherein#asexual#forms,#compared#to#their#sexual#relatives,#tend#to#have#broader#ranges,#colonize#higher#latitudes#and#altitudes,#and#generally#occupy#areas#of#the#range#opened#by#glacial#retreat#(Bierzychudek,#1985).##At#present,#no#data#exist#to#explain#why#the#geographic#distribution#of#apomicts#significantly#exceeds#that#of#diploid#populations#in#the#Easter#Daisy.#Likewise,#the#factors#that#allow#the#two#forms#to#persist#across#the#species’#range,#and#that#limit#their#co[occurrence#at#local#scales#are#unknown.#It#has#been#hypothesized#that#sexual#diploids#formerly#occupied#a#broad#range#that#was#disrupted#by#the#last#period#of#glaciation,#which#confined#some#relic#populations#to#the#southern#7"limit#of#the#original#species’#range.#Following#glacial#retreat,#apomictic#plants#were#more#successful#than#their#sexual#relatives#in#recolonizing#the#postglacial#landscape#(Thompson#and#Whitton,#2006).##1.5 Hypotheses(for(the(occurrence(of(sexual(and(apomictic(populations(in(Townsendia(hookeri(Although#probably#transient#over#evolutionary#time#scales#(e.g.,#Stebbins,#1950;#Maynard#Smith,#1978;#Van#Dijk,#2003),#the#success#of#apomictic#lineages#over#their#sexual#progenitors#is#remarkable#and#could#be#the#result#of#different#factors.#One#clear#advantage#of#apomixis#is#uniparental#reproduction,#which#guarantees#no#dependence#on#mating#partners#or#pollinators,#and#decreases#inbreeding#depression#through#maintenance#of#heterozygosity.#Reproductive#assurance#(Stebbins,#1950)#would#facilitate#the#establishment#of#apomicts#via#long[distance#dispersal#of#seeds#in#newly#opened#territory,#conferring#an#initial#benefit#to#polyploid#cytotypes#over#their#diploid#progenitors#(e.g.,#Hörandl,#2006).#Moreover,#asexual#polyploids#could#rely#on#a#more#plastic#genotype,#characterized#by#broad#tolerance#ranges,#which#allow#them#to#occupy#a#wider#spectrum#of#habitats#(“general#purpose#genotype#hypothesis”;#Lynch,#1984).#According#to#Lynch#(1984),#the#most#successful#clone#lineages,#in#the#long#term,#are#those#with#a#highly#generalized#genetic#makeup,#because#specialized#clones#will#disappear#as#soon#as#the#narrow#habitats#in#which#they#thrive#are#no#longer#available.#Broader#ecological#tolerances#(such#as,#for#instance#a#greater#resistance#to#pathogens,#drought#or#cold)#could#also#be#the#result#of#the#phenotypic#and#genetic#effects#associated#with#a#polyploid#condition#(Levin,#1983;#Otto#and#Whitton,#2000),#which#is#typical#of#apomictic#forms.##These#arguments#might#explain#why#apomictic#polyploids#are#able#to#establish#in#a#site#but#do#not#clarify#why#they#would#not#be#invaded#by#sexuals#in#the#long#term.#An#alternative#explanation#is#that#the#distribution#of#related#sexual#and#asexual#breeding#types#could#have#been#shaped#by#the#8"disruptive#effects#of#pollen#produced#by#apomicts#on#the#reproduction#of#their#outcrosser#relatives.#In#this#scenario,#whenever#sexual#individuals#disperse#into#an#area#occupied#by#their#apomictic#descendants#they#receive#primarily#pollen#of#polyploid#origin.#Despite#its#low#viability,#such#pollen#can#function#in#crosses#with#sexual#seed#parents,#as#shown#in#many#experimental#studies#(e.g.,#Sterk,#1987;#Morita#et.al.,#1990b;#Tas#and#Van#Dijk,#1999;#Noyes,#2000;#Hörandl#and#Temsch,#2009).#Sexual[asexual#crosses#can#have#many#effects#on#the#reproductive#performance#of#diploids.#For#instance,#pollen#from#apomicts#can#produce#a#decreased#seed#set#on#sexual#mothers,#as#described#for#plants#of#Taraxacum.japonicum#that#grow#in#the#presence#of#the#asexual#congener#T..officinale#(Takakura#et.al.,#2009;#Matsumoto#et.al.,#2010).#The#F1#generation#can#also#have#reduced#fitness#owing#to#“mentor#effects”,#a#phenomenon#that#determines#the#breakdown#of#self[incompatibility#systems#in#outcrossing#sexuals#when#the#receptive#stigma#is#saturated#with#a#mixture#of#apomictic#and#self[pollen#(Richards,#1997).#Lastly,#viable#pollen#produced#by#asexual#cytotypes#can#mediate#the#transfer#of#apomictic#genes#to#the#offspring#of#sexual#conspecifics#through#the#generation#of#fertile,#apomictically#reproducing#hybrids.#The#inheritance#of#apomixis#via#hybridization#has#been#established#in#a#number#of#artificial#crosses,#for#example,#in#Erigeron#(Noyes#and#Rieseberg,#2000)#and#Taraxacum#(Tas#and#Van#Dijk,#1999),#and#was#also#observed#in#natural#mixed#sexual[apomictic#populations#(Van#Dijk,#2007).#As#a#result,#there#is#a#high#chance#that#the#reproductive#success#of#sexual#plants#receiving#pollen#from#surrounding#apomicts#would#be#greatly#reduced#and#that,#eventually,#outcrossers#would#be#eliminated#entirely#from#the#local#mixed[population#(Mogie,#1992;#Mogie#et.al.,#2007).#Therefore,#this#unidirectional#interaction#(autonomous#apomicts#can#serve#as#pollen#parents#of#progeny#of#outcrossers,#but#not#vice#versa)#can#be#seen#as#a#form#of#reproductive#interference#(Kuno,#1992)#that#leads#to#the#creation#of#an#impenetrable#front#that#blocks#the#northward#migration#of#sexual#diploids#into#the#territory#occupied#by#their#polyploid#descendants.#Uniparental#reproduction,#the#general#purpose#genotype#hypothesis,#polyploidy#and#reproductive#interference#could#explain#why#apomictic#polyploid#individuals#prevail#in#most#of#the#species’#9"range,#but#these#hypotheses#do#not#clarify#why#apomicts#fail#to#thrive#in#areas#where#the#diploids#are#found#and#where#they#originated.#A#possible#explanation#is#that#outcrossing#diploids#are#restricted#to#a#portion#of#the#range#where#they#hold#a#selective#advantage#over#their#asexual#counterparts.#Indeed,#the#two#cytotypes#could#differ#in#ecological#traits#and,#in#particular,#they#could#have#divergent#edaphic#tolerances.#Beaman#(1957)#recognized#the#importance#of#edaphic#factors#in#influencing#the#distribution#of#the#species#of#Townsendia#and#claimed#that#“especially#within#the#range#of#a#species,#the#occurrence#of#plants#on#localized#edaphic#situations#is#frequently#striking.”#The#analysis#of#soil#samples#collected#at#different#localities#across#the#range#showed#that#the#concentration#of#some#minerals#(especially#Se,#Pb,#Al,#Cu,#Ni#and#Mg)#varies#among#different#habitats#(Thompson#and#Whitton,#unpublished),#and#it#has#been#suggested#that#diploids#may#occur#on#substrates#with#unusual#concentrations#of#specific#elements#(Whitton#et.al.,#2008).#1.6 Study(objectives(The#overall#objective#of#this#study#was#to#expand#our#understanding#of#the#factors#that#have#shaped#and#maintain#the#geographical#distribution#of#sexual#and#apomictic#populations#of#Townsendia#hookeri#across#the#species’#range.#The#following#broad#questions#were#addressed:##I. Why#are#sexual#populations#restricted#to#the#southern,#unglaciated#portions#of#the#species#range?##II. What#factors#limit#the#co[occurrence#of#sexual#and#apomictic#forms#at#local#scales?#The#focus#of#this#study#was#to#test#the#hypothesis#that#reproductive#interference#might#prevent#the#coexistence#of#different#breeding#types#within#a#population#and#hinder#the#range#expansion#of#sexuals#into#the#territory#occupied#by#their#apomictic#descendants.#To#test#this#hypothesis,#I#cross[pollinated#sexual#and#apomictic#populations#in#the#field,#I#assessed#the#reproductive#success#of#seed#paternal#plants#and#I#determined#whether#sexual[asexual#crosses#produced#F1#polyploid#hybrids. 10" Figure(1.1(Flowering#plant#of#Townsendia.hookeri.#The#picture#was#taken#in#the#Medicine#Bow#National#Forest#(Wyoming,#U.S.)#in#April#of#2012.# Figure(1.2(Plant#of#Townsendia.hookeri#at#fruiting#stage,#showing#a#typical#seed#head.#The#picture#was#taken#in#the#Medicine#Bow#National#Forest#(Wyoming,#U.S.)#in#June#of#2012.#11"Chapter(2: Could(reproductive(interference(limit(the(range(expansion(of(sexual(populations(of(Townsendia*hookeri*into(the(territory(occupied(by(their(polyploid(descendants?(2.1 Introduction(Townsendia#hookeri#(Asteraceae)#is#a#diminutive,#perennial#herbaceous#species#that#thrives#on#the#foothills#of#the#Rocky#Mountains,#from#central#Colorado#to#British#Columbia,#with#a#disjunct#distribution#in#Yukon#Territory#(Beaman,#1957;#Thompson#and#Whitton,#2006).#Outside#Yukon,#sexual#populations#are#only#found#in#the#southernmost#portion#of#the#species#range#(Colorado#and#southern#Wyoming),#whereas#apomictic#populations#occur#from#southern#Wyoming#to#Alberta#and#British#Columbia.#Moreover,#in#T.#hookeri#the#reproductive#strategy#has#been#shown#to#be#tightly#coupled#with#ploidy#level,#where#diploid#plants#are#invariably#outcrossers#and#polyploidy#is#always#associated#with#asexuality#in#the#form#of#apomixis#(Beaman,#1954;#Beaman,#1957).#Apomictic#forms#retain#the#ability#to#produce#showy#flowers#and#some#functional#pollen#despite#both#endosperm#and#embryo#development#are#completely#independent#of#pollination#(Beaman,#1954;#Beaman,#1957).##No#data#exist#to#explain#why#sexual#populations#fail#to#invade#the#territory#occupied#by#apomictic#populations,#and#why#the#two#forms#do#not#co[occur#at#local#scales.#This#study#focused#on#the#hypothesis#that#the#occurrence#of#asymmetric#reproductive#interference#between#apomictic#polyploid#and#sexual#diploid#cytotypes#might#have#the#potential#to#hinder#the#coexistence#of#different#breeding#types#at#local#scales#and#could#limit#the#northward#migration#of#sexuals.#To#test#this#hypothesis,#I#conducted#a#cross[pollination#experiment#between#extant#sexual#and#apomictic#populations#in#the#field.#To#properly#interpret#my#results,#I#collected#data#on#the#reproductive#success#of#sexual#and#asexual#plants#under#natural#conditions,#and#I#confirmed#apomixis#and#sexuality#using#a#pollinator#exclusion#test.#Reproductive#success#of#seed#mother#plants#was#12"assessed#based#on#seed#set,#achenes#mass,#germination#rate#and#seedling#survival.#Ploidy#of#seed#and#pollen#parents#was#confirmed#using#flow#cytometry#on#silica[dried#leaf#tissue#and#was#used#to#infer#breeding#system.#The#same#technique#was#applied#to#the#progeny#of#sexual[asexual#crosses#to#verify#the#occurrence#of#F1#polyploid#hybrids.#2.2 Methods(2.2.1 Study(site(In#his#monograph,#Beaman#(1957)#describes#Townsendia.hookeri#as#a#promising#but#also#challenging#study#system,#in#particular#because,#to#his#knowledge,#no#one#had#succeeded#in#getting#greenhouse[grown#plants#to#flower#and#set#seed.#After#over#fifty#years,#we#have#not#yet#been#able#to#overcome#this#major#obstacle,#therefore#I#did#my#experiment#in#the#field,#rather#than#working#in#a#more#conventional#and#controlled#environment,#such#as#a#greenhouse#or#growth#chamber.#To#conduct#the#field#experiment,#it#was#necessary#to#identify#six#natural#populations#(two#apomicts#and#four#sexuals)#each#with#at#least#fifty#flowering#individuals,#that#were#all#within#a#two#hour[drive#(less#than#80#km#apart)#so#that#I#could#collect#pollen#and#complete#crosses#within#a#single#day.#Six#populations#with#compatible#characteristics#were#located#in#southern#Wyoming#(Albany#county)#based#on#previous#work#(Thompson#and#Whitton,#2006;#Lee#and#Whitton,#unpublished#data).#These#populations#were#at#least#1#km#apart#from#each#other#and#they#were#all#found#in#the#Medicine#Bow#National#Forest,#with#the#exception#of#one#population#that#was#located#on#private#land#outside#the#city#of#Laramie#(Figure#2.1).#The#ploidy#level#and#breeding#system#of#each#population#was#subsequently#confirmed#using#pollen#analysis#(following#the#methods#described#in#Appendix#A)#and#flow#cytometry#on#silica[dried#leaf#tissue#(described#in#section#2.2.4).#A#summary#of#the#main#features#of#each#population,#including#geographical#locations,#IDs#and#ploidy#estimates#are#provided#in#Table#2.1.#13"2.2.2 Experimental(design(and(field(work(The#six#populations#of#T..hookeri#were#split#into#two#subsets,#corresponding#to#two#replicate#experiments#(R1#and#R2).#Each#subset#included:#one#apomictic#pollen#donor#population#(AD),#one#sexual#pollen#donor#population#(SD),#and#one#sexual#pollen#recipient#population#(SR).#Within#a#population,#individuals#were#randomly#assigned#to#a#specific#treatment,#and#only#one#flower#head#per#plant#was#used.#This#approach#avoided#pseudoreplication,#while#reducing#the#impact#to#individual#plants.#Flower#heads#were#enclosed#with#semi[transparent#fine[mesh#organza#bags#that#prevented#achene#dispersal#and#hampered#animal#and#wind#pollination#(Cosendai#et.al.,#2013).#Flower#heads#were#exposed#to#one#of#four#different#treatments:#control#(C),#isolation#(I),#sexual[sexual#crossing#(SS)#or#sexual[apomictic#crossing#(SA),#as#shown#in#Figure#2.2.#The#control#was#used#to#assess#seed#production#under#open#pollination.#Flower#heads,#with#withered#ray#florets#and#developing#infructescences,#were#bagged#to#prevent#wind#dispersal#of#achenes.#The#control#was#performed#on#ten#plants#in#both#apomictic#(AD)#and#sexual#(SR)#populations,#for#a#total#of#forty#flower#heads.#The#isolation#treatment#was#used#to#confirm#that#polyploids#are#apomicts#and#diploids#are#self[incompatible#outcrossers.#In#a#closely#related#species,#Townsendia.aprica,#Tepedino#et.al.#(2004)#compared#the#effects#of#autogamy#(isolated#and#unmanipulated#flower#heads)#versus#geitogamy#(receptive#stigmas#treated#with#fresh#pollen#from#other#florets#on#the#same#plant)#and#found#no#significant#difference#between#the#two#treatments,#which#both#produced#low#seed#sets#(<2.5%#of#disk#florets#produced#filled#achenes).#Based#on#these#results,#very#low#seed#sets#in#isolated#sexual#plants#are#considered#an#indication#of#self[incompatibility,#although#a#definitive#test#would#require#the#application#of#self[pollen.#As#with#the#controls,#the#isolation#treatment#was#carried#out#on#ten#plants#in#both#the#two#AD#and#the#two#SR#populations#(forty#individuals#in#total),#and#involved#bagging#flower#heads#at#the#bud#stage#to#prevent#them#from#receiving#any#foreign#pollen.#It#should#be#noted#that#self[pollen#was#not#removed#because#emasculation#of#florets#has#not#been#accomplished#in#this#system#(in#the#Asteraceae,#this#is#sometimes#done#by#cutting#off#the#top#of#14"each#inflorescence#when#in#bud;#Richards,#1997),#and#it#was#also#not#pivotal#to#the#isolation#test.#The#same#method#has#been#successfully#applied#to#other#Compositae,#where#the#removal#of#anthers#was#similarly#not#practical#(e.g.,#Morita#et.al.,#1990b;#Noyes,#2000;#Brock,#2004).#The#goal#of#sexual[sexual#and#sexual[apomictic#crosses#was#to#compare#the#effects#of#conspecific#(from#diploids)#versus#heterospecific#(from#polyploids)#pollen#grains#on#reproductive#success#of#sexual#seed#parents#under#hand[pollination.#For#this#reason,#the#two#treatments#only#differed#in#the#source#of#pollen#grains,#which#was#either#sexual#(from#SD#population,#in#SS)#or#apomictic#(from#AD#population,#in#SA).#The#crosses#were#performed#by#gently#rubbing#two#flower#heads#from#a#donor#population#on#the#flower#head#of#a#sexual#recipient#during#anthesis.#Each#donor#inflorescence#was#collected#from#a#different#plant#and#used#only#once#to#avoid#pollen#contamination#between#crosses.#In#each#recipient#population,#fifty#flower#heads#(one#hundred#in#total)#were#bagged#before#the#onset#of#anthesis#and#assigned#to#one#of#the#two#treatments,#so#that#twenty[five#individuals#received#pollen#from#a#diploid#donor#and#the#other#twenty[five#received#pollen#from#a#polyploid#donor.#Bags#were#opened#only#to#perform#the#artificial#pollination.#Flower#heads#from#pollen#donor#populations#were#collected,#kept#in#paper#bags#and#stored#at#about#4˚C#for#less#than#two#hours#before#the#hand[pollination#was#completed.##The#treatments#were#conducted#in#April#2012#and#achenes#were#collected#in#June#2012,#with#the#exception#of#the#isolation#treatment#on#apomicts,#which#was#conducted#in#2013,#because#I#could#not#find#a#sufficient#number#of#buds#during#the#first#year.#Moreover,#population#2027#was#severely#affected#by#a#wildfire#during#the#summer#of#2012#and#it#was#necessary#to#conduct#the#isolation#treatment#on#a#different#population.#Population#2031,#previously#described#by#Lee#and#Whitton#(unpublished#data),#was#determined#to#be#an#adequate#replacement#based#on#its#location#(roughly#20#km#north#of#Laramie)#and#ploidy#level#(polyploid).#Subsequent#analyses#confirmed#that#pollen#viability#and#ploidy#level#of#members#of#this#population#are#comparable#to#those#of#population#2027#(Table#2.1,#Figure#2.1).#Hand[pollination#was#repeated#twice#over#a#three[day#period#in#population#15"2030.#This#was#not#achieved#for#population#2033,#due#to#weather#conditions#(snow#covering#the#plants);#therefore#recipients#in#this#population#received#pollen#only#once.#An#attempt#to#repeat#the#entire#experiment#in#2013#was#impeded#by#adverse#weather#conditions#and#consecutive#snow#storms.#Thus,#the#two#experiments#are#not#considered#replicates#as#originally#intended,#and#were#analyzed#separately,#comparing#the#effects#of#two#versus#one#day#of#cross[pollination.#Achenes#were#retrieved#from#176#of#the#180#plants#bagged#under#one#of#the#four#treatments.#Only#four#individuals#could#not#be#relocated,#thus#the#organza#bags#proved#to#be#highly#effective#at#preventing#seed#dispersal,#without#hindering#the#survival#of#the#plant.#A#summary#of#the#work#completed#in#the#field#is#provided#in#Table#2.2.##2.2.3 Estimation(of(reproductive(success(The#reproductive#success#of#seed#mother#plants#under#the#four#treatments#was#assessed#by#evaluating#the#cumulative#effects#of#four#different#parameters#associated#with#achenes#quantity#and#quality,#namely:#seed#set,#average#achene#mass,#germination#rate#and#seedling#survival.##The#main#goal#of#the#cross[pollination#experiment#was#to#test#the#potential#effects#of#pollen#produced#by#apomicts#on#the#reproductive#success#of#sexual#seed#parents.#To#understand#the#magnitude#of#this#interaction,#I#first#needed#to#gather#information#on#the#reproductive#success#of#both#cytotypes#under#natural#conditions,#i.e.#the#control#treatment.#I#also#had#to#show#that#the#absence#of#external#pollen#(in#the#isolation#treatment)#would#not#affect#reproductive#success#of#polyploids#(consistent#with#their#being#apomictic),#but#would#drastically#impact#diploids,#demonstrating#that#the#latter#are#strictly#sexual#outcrossers.#For#this#reason,#seed#set,#mass#of#achenes,#germination#and#seedling#survival#rate#were#initially#measured#and#compared#among#cytotypes#under#control#and#isolation#treatments,#and#secondly#among#cross[pollination#treatments#in#sexual#populations.#16"2.2.3.1 Seed(set(Seed#heads#(capitula)#retrieved#from#the#field#were#kept#in#paper#bags#and#stored#at#4˚C#inside#a#box#containing#silica#gel.#The#total#number#of#achenes#collected#from#each#seed#head#was#recorded,#and#achenes#were#classified#as#“filled”,#when#plump#and#brown,#and#as#“empty”,#when#flat#and#unpigmented.#Fruits#with#intermediate#colorations#were#scored#as#filled#if#fleshy#and#as#empty#if#clearly#undeveloped#(Figure#2.3).##Seed#set#was#calculated#as#the#proportion#of#filled#achenes#in#a#given#seed#head,#and#it#was#expressed#as#a#percentage:##Seed!set! % = ! no. filled!achenesno. filled + no. empty!achenes! ×!100#2.2.3.2 Average(achene(mass((mg)(The#average#mass#of#filled#achenes#was#calculated#for#individual#seed#heads#by#averaging#the#weight#of#the#entire#set#of#fruits.#The#estimates#are#reported#in#milligrams.#(2.2.3.3 Germination(rate((Germination#tests#were#conducted#on#a#random#subset#of#filled#and#empty#achenes#obtained#from#every#bagged#plant#that#produced#fruits#under#one#of#the#four#treatments.#During#preliminary#trials,#I#noticed#that#undeveloped#fruits#are#more#susceptible#to#fungal#and#bacterial#infections,#therefore#I#decided#to#germinate#the#two#achene#types#following#the#same#protocol,#but#separately.##The#seeds#of#T..hookeri#do#not#possess#dormancy,#therefore#no#stratification#or#any#other#form#of#conditioning#is#necessary#to#induce#germination#(personal#observation).#Ten#achenes#(or#all#the#achenes#if#fewer#were#available)#were#randomly#selected#from#each#maternal#plant.#After#the#pappus#was#carefully#cut#off,#achenes#were#surface#sterilized#with#a#10%#(v:v)#bleach#solution#for#5#17"minutes#and#rinsed#three#times#with#distilled#water.#A#1%#(w:v)#agar#solution#was#autoclaved#and#poured#into#sterile#100#×#15#mm#Petri#dishes#in#a#laminar#flow#hood.#Once#the#medium#solidified,#the#sterilized#achenes#from#each#maternal#plant#were#sown#in#two#different#Petri#plates,#so#that#the#insurgence#of#any#infection#inside#a#dish#would#affect#and#potentially#kill#only#half#or#less#of#the#fruits#of#a#single#plant,#reducing#the#risk#of#losing#entire#subsets.#Petri#plates#were#sealed#with#parafilm#and#transferred#to#a#growth#chamber#(Adaptis#A1000,#Conviron,#Winnipeg,#MB,#Canada),#where#achenes#were#incubated#at#a#constant#temperature#of#23˚C,#with#12#hours#of#light#and#12#hours#of#dark#per#day.#Achenes#were#inspected#every#two#days#over#the#course#of#three#weeks,#and#recorded#as#germinated#once#the#radicle#was#at#least#5#mm#long.##The#germination#rate#was#calculated#as#the#proportion#of#sown#fruits#(filled#or#empty)#that#successfully#germinated,#and#it#was#reported#as#a#percentage.#For#instance,#the#germination#rate#of#filled#achenes#for#a#given#plant#was#calculated#as:#Germination!rate!per!filled!achene! % = !no. filled!achenes!germinated!no. filled!achenes!sown! ! ×!100 2.2.3.4 Survival(rate(per(transplanted(seedling(All#seedlings#obtained#from#C#and#SA#treatments#were#transferred#from#agar#to#soil,#whereas#only#a#fraction#of#the#seedlings#obtained#from#SS#treatment#(representing#eight#mother#plants#per#population)#and#none#from#the#I#treatment#were#potted#because#of#space#constraints.##The#seedlings#were#removed#from#the#plate#together#with#a#small#amount#of#agar#still#surrounding#the#tap#root#in#order#to#minimize#the#risk#of#damaging#or#breaking#the#radicles.#After#the#transplant,#seedlings#were#kept#in#a#growth#chamber#(Bigfoot#series,#model#GC[20,#BioChambers#Inc.,#Winnipeg,#MB,#Canada)#and#exposed#to#a#daily#regime#of#12#hours#of#light#at#20˚C#followed#by#12#hours#of#dark#at#10˚C.##18"I#observed#that,#at#the#stage#of#four#to#five#true#leaves,#seedlings#had#already#developed#an#extensive#tap#root#(Figure#2.4),#and#could#be#considered#fully#established#plants.#Based#on#this#observation,#the#survival#rate#was#calculated#as#the#percentage#of#transplanted#seedlings#that#developed#a#minimum#of#four#true#leaves:#Survival!rate!per!transplanted!seedling! % = ! no. established!plants!no. transplanted!seedlings!! ×!100#2.2.3.5 Statistical(analysis(Seed#set,#achene#mass,#germination#and#survival#rate#were#computed#for#each#seed#mother#plant,#as#described#above.#Comparisons#of#seed#set,#germination#rate#and#survival#rate#between#treatments#and#breeding#systems#were#evaluated#using#a#generalized#linear#model#(GLM)#by#performing#an#Analysis#of#Deviance#with#binomial#errors#(and#a#logit#link#function).#This#method#was#selected#following#Crawley#(2007),#who#showed#that#the#‘glm’#function#represents#an#adequate#tool#for#the#analysis#of#proportion#data,#and#can#replace#more#traditional#approaches,#such#as#arcsine#transformation#or#non[parametric#statistics.#The#differences#in#average#mass#of#filled#achenes#between#treatments#and#breeding#systems#were#tested#using#Student's#t[tests.#The#statistical#analysis#was#carried#out#in#R#(version.#3.0.3;#R#Core#Development#Team,#2014)#using#the#“lattice”#package#for#data#visualization#(Sarkar,#2008)#and#the#“plyr”#and#“car”#packages#for#data#manipulation#(Fox#and#Weisberg,#2011;#Wickham,#2011).#(19"2.2.4 Flow(cytometry((FCM)(2.2.4.1 Plant(material((Flow#cytometry#was#used#to#confirm#the#ploidy#and#breeding#system#of#pollen#and#seed#parental#plants,#previously#inferred#via#pollen#analysis#(following#the#methods#reported#in#Appendix#A),#and#to#assess#ploidy#level#of#the#progeny#generated#in#sexual[asexual#crosses#(SA#treatment#).#The#ploidy#level#of#putative#apomictic#pollen#donor#and#diploid#pollen#recipient#plants#was#estimated#from#field[collected#tissue#(samples#from#sixteen#individuals#harvested#in#June#2012)#as#well#as#from#material#grown#from#seeds#(samples#from#seventeen#individuals).#The#latter#was#obtained#from#achenes#germinated#and#grown#in#a#growth#chamber,#following#the#protocol#described#previously.#These#achenes#were#harvested#from#control#plants,#therefore#they#are#representative#of#the#breeding#system#of#their#population#of#provenance.#Leaf#material#of#both#origins#(field[collected#and#grown#from#seed)#was#stored#at#ambient#temperature#in#paper#envelopes#containing#silica#gel.#Within#one#to#four#weeks#of#collection,#the#dehydrated#tissue#was#removed#from#the#silica,#transferred#to#2#ml#screw#cap#microtubes#and#stored#in#a#[80˚C#freezer.#The#rationale#for#analyzing#desiccated#material#instead#of#fresh#leaves#was#to#ensure#that#the#results#were#comparable#to#the#ploidy#assessments#carried#out#by#Garani#et.al.#(unpublished)#on#field[collected,#silica[dried#leaf#tissue#gathered#from#populations#across#most#of#T.#hookeri’s#range#(Appendix#B).#Achenes#produced#by#seed#parents#under#the#SA#treatment#were#germinated#in#February#2013#and#seedlings#were#grown#as#described#above#until#large#enough#to#allow#sampling#of#leaf#material.#Between#December#2013#and#January#2014,#20#to#40#mg#of#fresh#tissue#(ten#to#twenty#leaves)#were#collected#from#mature#plants.#Leaf#material#was#harvested#only#from#large#individuals#that#had#at#least#twice#as#many#leaves#(177#in#total)#to#avoid#killing#the#plants.#This#method#ensured#that#material#could#be#collected#multiple#times#from#a#single#plant,#allowing#for#a#reassessment#of#low#20"quality#results.#Leaf#tissue#was#desiccated#in#silica#gel#and#stored#in#a#deep#freezer#([80˚C),#as#previously#described#for#pollen#and#seed#parents.#2.2.4.2 PloidyYlevel(determination((For#sample#preparation,#10#mg#of#dried#leaf#tissue#was#placed#in#a#2#ml#microcentrifuge#tube#containing#a#6.35#mm#ceramic#sphere#(Qbiogene,#catalog#no.#6540[424)#and#600#µl#of#nuclear#isolation#buffer#(pH#7.0)#of#the#following#composition:#15#mM#HEPES,#1#mM#EDTA,#0.2%#(v:v)#Triton#X[100,#80#mM#KCl,#20#mM#NaCl,#300#mM#sucrose,#0.5#mM#spermine,#0.25#mM#PVP[40#(modified#from#De#Laat#and#Blaas,#1984),#plus#1#µl#ml[1#ß[mercaptoethanol.#The#tubes#were#placed#symmetrically#into#a#test#tube#rack,#modified#to#attach#to#a#Mastercraft#7.5A#reciprocating#saw#(with#a#stroke#length#of#28.6#mm),#and#the#leaf#material#was#ground#by#oscillating#the#tubes#on#the#saw#at#‘low’#setting#for#60#seconds.#This#method#for#extraction#of#plant#material#by#using#a#reciprocating#saw#has#been#shown#to#be#highly#effective#and#reliable#(Alexander#et.al.,#2007),#as#it#ensures#higher#consistency#and#is#less#labor#intensive#compared#to#chopping#samples#with#a#razor#blade#(Galbraith#et.al.,#1983;#Doležel#et.al.,#2007).#The#resulting#homogenate#was#passed#through#a#Partec#CellTrics®#columns#equipped#with#a#30#µm#filter#gauze#(Münster,#Germany)#and#collected#into#1.5#ml#centrifuge#tubes.#Before#and#after#filtering#the#homogenate,#50#µl#of#nuclear#isolation#buffer#was#added#to#each#column#to#pre[wet#the#filter#and#then#to#remove#any#residual#nuclei#from#the#gauze.#The#final#volume#of#filtrate#in#each#1.5#ml#tube#was#approximately#700#µl.#The#nuclei#suspensions#were#stored#in#ice#for#45#to#60#minutes#and#then#moved#to#a#4˚C#refrigerator#for#up#to#three#hours#prior#to#running#on#the#flow#cytometer.##Pisum.sativum.cv.#Ctirad#(2C[value#9.09#pg;#Doležel#et.al.,#1998),#Secale.cereale.cv.#Daňkovské#(2C[value#16.19#pg;#Doležel#et.al.,#1998),#and#Vicia.faba.cv.#Inovec#(2C[value#26.9#pg;#Doležel#et.al.,#1992)#were#chosen#as#DNA#standards.#For#each#species,#two#to#three#plants#were#grown#from#seed#and#fully#expanded#leaves#were#harvested,#desiccated#using#silica#gel#and#stored#in#a#deep#freezer#21"(at#[80˚C),#following#the#same#method#applied#to#T..hookeri#samples.#Nuclei#suspensions#were#prepared#as#described#above.#When#using#an#internal#reference,#it#is#recommended#the#standard#be#ground#with#each#sample#(Price#et.al.,#2000).#At#the#same#time,#it#is#necessary#to#ensure#that#the#genome#size#of#the#sample#is#as#close#as#possible#to#the#genome#size#of#the#standard,#never#exceeding#a#twofold#difference#and#avoiding#overlap#(Suda#and#Trávníček,#2006;#Doležel#et.al.,#2007).#The#DNA#content#of#offspring#from#SA#treatment#was#unknown#and#could#range#from#diploid#(roughly#12#pg)#to#tetraploid#(over#21#pg),#thus#no#internal#standard#was#adequate#for#the#whole#range#of#genome#sizes;#this#is#the#reason#references#were#prepared#separately.#Nonetheless,#following#recommendations#of#Price#et.al..(2000),#a#subset#of#samples#of#known#genome#size#was#ground#together#with#the#appropriate#internal#standard#to#test#for#the#presence#of#cytosolic#inhibitors#and#ensure#that#independently#processing#target#and#reference#leaf#tissue#does#not#alter#the#outcome#of#the#analysis.#The#2C[values#obtained#in#the#co[ground#trial#matched#the#ploidy#level#previously#estimated#for#the#population#of#origin#of#each#sample#(Appendix#C),#providing#evidence#that#my#method#produces#accurate#results#in#this#study#system.##The#nuclei#suspensions#were#incubated#with#RNase#A#(50#μg#ml[1;#Invitrogen)#for#30#minutes#at#room#temperature#prior#to#being#analyzed#on#a#Becton[Dickinson#FACScalibur#(BD,#San#Jose,#CA,#USA)#benchtop#flow#cytometer#equipped#with#a#488#nm#laser,#following#the#procedure#presented#in#Sears#(2011).#Data#were#acquired#and#analyzed#using#BD#CellQuest#Pro#software#(BD,#1#Becton#Drive,#Franklin#Lakes,#New#Jersey,#USA#07417),#which#also#allowed#adjustment#of#the#settings#of#the#cytometer.#At#first,#an#aliquot#of#each#internal#standard#was#stained#with#100#μg#ml[1#of#propidium#iodide#(PI,#Invitrogen)#and#run#on#the#cytometer#on#its#own#to#ensure#that#the#sample#preparation#yielded#good#quality#nuclei#extractions.#In#addition,#the#voltage#of#the#photomultipliers#(FL2)#was#adjusted#so#that#the#peak#of#the#G0/G1#phase#nuclei#of#P..sativum.occurred#at#channel#number#200#on#the#FL2[A#(“area”,#a#measure#of#relative#florescence)#axis.#G0/G1#and#G2#populations#of#single#nuclei#were#gated#(i.e.,#isolated)#on#the#plot#that#shows#pulse#width#(FL2[W)#against#area#(FL2[A)#and#the#22"rate#at#which#the#G0/G1#peak#of#each#standard#increased#on#the#histogram#of#fluorescence#intensities#(FL2[A#histogram)#was#recorded.#Aliquots#of#each#T..hookeri#sample#were#stained#and#run#following#the#same#procedure.#This#method#allowed#identification#of#the#proper#internal#standard#for#each#sample,#so#that#reference#and#target#tissue#G0/G1#populations#of#nuclei#were#as#close#as#possible#on#the#FL2[A#axis,#without#overlapping.#The#ratio#of#internal#standard#to#sample#was#determined#based#on#the#relative#rate#of#increase#of#each#G0/G1#peak#on#the#FL2[A#histogram#previously#measured,#with#the#intent#to#produce#two#G0/G1#peaks#of#similar#height.#Reference#and#T..hookeri#samples#were#combined#accordingly,#co[stained#and#run#twice,#until#a#minimum#of#2,000#particles#were#measured#in#every#individual#run.#For#example,#P..sativum#always#produced#a#rapidly#growing#G0/G1#peak#on#the#FL2[A#histogram#when#run#on#its#own,#while#diploid#T..hookeri#samples#were#generally#four#times#slower.#In#this#case,#20#µl#of#P..sativum.would#be#mixed#with#80#µl#of#T..hookeri#and#5#µl#of#PI.#For#the#subset#of#samples#of#T..hookeri#of#known#genome#size#that#were#ground#together#with#the#internal#standard,#100#µl#of#the#mixed#nuclei#suspension#was#stained#with#5#µl#of#PI#and#run#following#the#same#protocol#outlined#previously.#The#files#produced#in#BD#CellQuest#were#analyzed#using#the#computer#program#FlowJo#vX.0.6#(Tree#Star#Inc.,#Ashland,#Oregon,#USA).#The#fluorescence#of#the#sample#relative#to#the#internal#standard#was#determined#based#on#the#FL2[W#versus#FL2[A#cytogram#obtained#for#each#individual#run.#On#this#plot,#G0/G1#populations#of#nuclei#from#both#the#reference#and#the#target#tissue#were#isolated#from#the#debris#background#using#the#Autogate#feature,#which#automatically#finds#subpopulations#of#particles#based#upon#equal#probability#distribution#(Figure#2.5).#FlowJo#was#also#used#to#calculate#the#total#number#of#nuclei,#the#mean#position#(channel#number,#CN)#and#the#coefficient#of#variation#(CV,#defined#as#the#standard#deviation#of#the#peak#divided#by#its#mean#channel#number#and#expressed#as#percentage)#of#each#peak#on#the#FL2[A#histogram.#In#accordance#with#Suda#and#Trávníček#(2006),#the#outcome#of#an#individual#run#was#included#in#the#analysis#when#the#following#quality#requirements#were#met:#the#fluorescence#intensities#for#at#least#2,000#nuclei#were#collected,#23"the#CV#values#of#the#reference#and#the#T..hookeri#sample#were#≤6%,#and#both#peaks#on#the#histogram#of#fluorescence#intensities#had#a#normal#shape.#The#genome#size#of#T..hookeri#samples#(2C[value)#were#calculated#based#on#the#linear#relationship#between#the#standard#and#the#sample#fluorescence#intensities,#as#follows:#2C!!. ℎ!!"#$%!sample! pg = !2C!internal!standard! pg !×!CN!!. ℎ!!"#$%!sampleCN!internal!standard! #Flow#cytometry#was#completed#between#December#2013#and#March#2014.#Calculations#and#graphical#analyses#of#the#data#were#conducted#using#the#“lattice”#package#(Sarkar,#2008#)#in#R#(version.#3.0.3;#R#Core#Development#Team,#2014).#2.3 Results(2.3.1 Reproductive(success(of(sexual(versus(apomictic(populations(under(natural(conditions((C(and(I(treatments)(For#this#analysis,#populations#of#the#same#cytotype#were#considered#replicates#and,#as#a#consequence,#were#grouped#together#as#‘sexuals’#(2030#and#2033)#versus#‘apomicts’#(2019,#2027#and#2031).#The#total#number#of#filled#and#empty#achenes#per#capitulum#was#consistent#across#the#four#populations,#with#an#overall#mean#and#standard#error#(SE)#of#81.21#±#3.79#achenes#per#capitulum#(Figure#2.6).#This#suggested#that#sexual#and#asexual#plants#used#in#this#study#had#flower#heads#of#similar#sizes,#based#on#the#assumption#that#the#total#number#of#achenes#can#be#used#as#a#proxy#of#the#inflorescence#size#because#each#floret#produces#only#one#ovule,#and#thus#one#fruit.##Under#the#control#treatment#sexuals#and#apomicts#had#similar#percentage#seed#set,#with#average#values#and#SE#of#48.96#±#3.15%#and#52.63#±#2.2%,#respectively#(Figure#2.7).#In#contrast,#the#24!isolation(treatment(drastically(decreased(the(percentage(of(developed(achenes(per(seed(head(produced(by(sexuals((0.84(±(0.34%),(but(had(no(significant(impact(on(apomicts((46.15(±(2.34%)(relative(to(controls((Analysis(of(Deviance,(P<2.2eI16;(Table(2.3).(All(apomictic(plants(in(this(experiment((n=38)(set(seed,(regardless(of(treatment,(and(the(same(was(true(for(openIpollinated(sexual(diploids((n=20).(Conversely,(twelve(out(of(nineteen(diploid(mothers(set(no(seed(in(the(isolation(treatment,(and(the(remaining(seven(produced(only(between(one(and(four(filled(achenes.(Although(polyploids(had(a(slightly(lower(seed(set(when(external(pollen(was(removed(compared(to(the(control(treatment,(most(of(the(offspring(produced(by(polyploids(under(isolation(treatment(were(viable((215(of(217(seeds(from(twentyItwo(parents(sown(in(agar(germinated).(As(observed(for(seed(set,(the(average(mass(of(filled(achenes(did(not(differ(significantly(between(the(two(mating(types(under(the(control(treatment,(with(a(mean(and(SE(of(0.69(±(0.033(mg(for(sexuals(and(0.77(±(0.027(mg(for(apomicts((t=1.8393,(df=36,(P=0.0741).(As(expected,(all(the(1560(empty(achenes(tested((representing(156(mother(plants)(failed(to(germinate.(The(average(germination(rate(per(filled(achene(was(significantly(lower(in(polyploid(controls((75.98(±(5.66%)(compared(to(diploid(controls((90.05(±(3.48%;(Analysis(of(Deviance,(P=0.0421;(Figure(2.8;(Table(2.4).(Conversely,(seedling(survival(rate(was(significantly(higher(for(apomictic(controls((91.11(±(2.53%)(than(for(sexuals((81.38(±(5.53%;(Analysis(of(Deviance,(P=0.0428;(Figure(2.9;(Table(2.5).((2.3.2 Experimental/crossings/(SS/and/SA/treatments)/Data(on(percentage(seed(set((Figure(2.10)(reveal(that(both(populations(were(pollen(limited(in(the(experimental(crosses,(with(over(a(60%(decline(in(seed(set(between(open(pollination((C)(and(crossing(with(conspecific(pollen((SS;(mean(and(standard(error(of(18.02(±(2.26%(for(2030(and(13.46(±(2.24%(for(2033).(Nonetheless,(the(experimental(crosses(were(effective(and(most(sexual(mother(plants(set(seed.(In(population(2030,(the(seed(set(of(plants(that(received(pollen(from(apomictic(donors(was(roughly(half(of(that(produced(by(individuals(that(received(pollen(from(sexual(donors(25!(10.34(±(1.5%(in(SA(and(18.02(±(2.26%(in(SS;(Analysis(of(Deviance,(P=0.004279;(Table(2.6),(and(only(one(plant((in(the(SA(treatment)(failed(to(set(seed.(In(population(2033,(the(average(seed(set(was(similar(in(the(two(treatments((15.25(±(3.41%(in(SA(and(13.46(±(2.24%(in(SS),(but(the(mean(values(for(seed(parents(in(SA(showed(a(great(degree(of(variation,(where(some(plants(failed(to(set(seed(entirely((n=8),(and(others((n=6)(had(high(seed(sets,(between(31%(and(50%.(The(average(mass(of(filled(achenes(was(significantly(different(between(the(two(recipient(populations((2030(had(larger(fruits),(but(not(between(treatments(within(a(population((Figure(2.11).(Nonetheless,(it(should(be(noted(that(achenes(from(sexualIasexual(crosses(in(population(2033(were(15%(lighter(than(those(from(sexualIsexual(crosses((0.47(±(0.03(mg(for(SA(versus(0.557(±(0.03(mg(for(SS).(In(contrast(to(seed(set,(the(germination(rate(was(not(affected(by(treatment(in(population(2030((Figure(2.12),(but(in(2033(mean(germination(was(significantly(lower(when(plants(received(apomictic((76.83(±(7.29%)(rather(than(sexual(pollen((85.87(±(5.11%)((Analysis(of(Deviance,(P=0.01492;(Table(2.7).(Finally,(Figure(2.13(shows(that(seedling(survival(rate(had(a(similar(trend(in(the(two(recipient(populations(in(response(to(treatment.(On(average,(offspring(of(sexualIapomictic(crosses(were(less(likely(to(survive(than(seedlings(from(sexualIsexual(crosses(and(the(decline(between(SS(and(SA(treatment(was(significant(in(both(populations((Analysis(of(Deviance,(P=0.035(for(2030(and(P=0.0129(for(2033;(Table(2.8).(2.3.3 Flow/cytometry/(FCM)/Of(the(434(achenes(from(sexualIasexual(crosses(that(germinated((221(from(22(mothers(in(population(2030(and(213(from(16(mothers(in(population(2033),(306(became(established(plants((i.e.,(survived).(It(should(be(noticed(that(the(majority(of(the(seedlings(that(perished(failed(to(develop(26"beyond#the#cotyledon#stage#once#moved#from#agar#to#soil.#FCM#was#conducted#on#a#subset#of#102#individuals,#selected#based#on#their#size#(smaller#plants#were#not#analyzed#because#they#did#not#provide#enough#leaf#material,#as#discussed#in#section#2.2.4.1)#and#representing#the#largest#number#of#maternal#plants#possible#(twenty#mothers#in#population#2030#and#twelve#mothers#in#population#2033).##Over#76%#of#the#samples#met#the#quality#standards#set#for#this#study#(26/33#for#parents#and#78/102#for#SA#treatment#progeny),#and#these#requirements#were#often#exceeded.#On#average,#I#was#able#to#detect#the#florescence#intensities#of#roughly#6,000#nuclei#per#sample,#and#for#most#measurements#the#CV#values#were#consistently#below#5%#(66.7%#of#observations#for#SA#progeny#and#80.8%#for#parents).#The#best#results#were#obtained#when#the#leaf#material#was#dried#in#silica#gel#for#21#days#or#longer,#and#polyploid#samples#typically#had#lower#nuclei#yields#and#higher#CVs,#as#previously#reported#by#Suda#and#Trávníček#(2006).#The#size#ranges#of#nuclear#DNA#corresponding#to#different#ploidies#of#T..hookeri#were#estimated#in#a#previous#work#by#performing#flow#cytometry#on#68#samples#collected#from#25#populations#known#to#be#either#diploid#or#polyploid#based#on#pollen#analysis#(Garani#and#Whitton,#unpublished;#see#Appendix#B).#These#estimates#largely#overlap#with#the#results#of#an#earlier#study#on#populations#of#T..hookeri#from#Yukon#Territory#(Thompson#et.al.,#2008).#Diploid#genome#size#was#between#11#and#14#pg,#whereas#polyploid#nuclear#DNA#formed#two#distinct#clusters:#genomes#between#17#and#21#pg#were#estimated#to#be#triploid#and#genomes#larger#than#21#pg#were#considered#to#be#tetraploid.#Based#on#this#method,#I#was#able#to#confirm#that#the#two#seed#parent#populations#(2030#and#2033)#were#diploid#and#the#two#apomictic#pollen#parent#populations#(2019#and#2027)#were#triploid#(Figure#2.14).#Furthermore,#flow#cytometry#data#showed#that,#as#expected,#individuals#in#population#2031#have#nuclear#DNA#sizes#comparable#to#those#of#plants#from#population#2027#(Figure#2.14).##27!The$analysis$of$leaf$tissue$from$the$offspring$of$sexual4apomictic$crosses$revealed$different$trends$in$the$two$recipient$populations$(Figure$2.15).$Roughly$half$of$the$thirty4seven$SA$offspring$from$2033$were$diploid$(48.7%)$and$only$one$plant$was$clearly$a$polyploid$(likely$a$triploid).$The$remaining$individuals$had$intermediate$nuclear$DNA$contents,$either$smaller$than$a$diploid$(2C4value$<$11$pg;$13.5%$of$the$samples),$or$larger$than$a$diploid$but$smaller$than$a$polyploid$genome$(14$pg$<$2C4value$<$17$pg;$35.1%$of$the$samples),$suggestive$of$an$aneuploid$condition.$In$contrast,$nearly$half$of$the$progeny$of$population$2030$was$found$to$be$polyploid$(twenty$out$of$forty4one),$with$a$striking$predominance$of$tetraploid$individuals.$Sixteen$plants$(39%)$were$clearly$diploid$and$five$(12.2%)$were$classified$as$aneuploid$(or$near4diploid),$because$their$genome$sizes$slightly$exceeded$the$upper$boundary$of$the$diploid$range$(2C4value$>$14$pg). 2.4 Discussion+In$this$study$we$showed,$for$the$first$time,$evidence$for$the$potential$existence$of$reproductive$interference$between$sexual$and$apomictic$populations$of$the$Easter$Daisy.$In$fact,$the$results$from$the$cross4pollination$experiment$indicated$that$pollen$produced$by$apomicts$could$reduce$the$reproductive$success$of$sexual$seed$mothers.$In$addition,$an$analysis$of$ploidy$level$revealed$the$presence$of$polyploid$cytotypes$within$the$F1$generation$obtained$from$sexual4asexual$crosses.$Thus,$pollen$from$apomictic$donors$certainly$sired$at$least$a$fraction$of$these$progeny.$These$findings$suggest$that$unidirectional$reproductive$interactions$mediated$by$pollen$might$limit$the$range$expansion$of$diploid$cytotypes$into$the$territory$occupied$by$their$polyploid$descendants.$My$experiment$also$showed$that$outcrossing$and$apomictic$cytotypes$were$found$to$have$comparable$reproductive$success$under$open$pollination,$at$least$in$the$southern$portion$of$the$species’$range,$where$the$experiment$was$conducted.$As$expected,$the$pollinator$exclusion$test$confirmed$apomixis$in$polyploid$populations$and$sexuality$in$diploid$populations.$$28"2.4.1 Reproductive(success(of(sexual(versus(apomictic(populations(under(natural(conditions((C(and(I(treatments)(The#isolation#treatment#showed#that#sexual#diploids#largely#failed#to#set#seed#when#florets#were#prevented#from#receiving#external#pollen.#Nonetheless,#a#few#mothers#produced#a#low#number#of#filled#achenes,#and#this#is#likely#due#to#accidental#pollen#contamination,#as#reported#in#similar#pollinator#exclusion#experiments#(O’Connell#and#Eckert,#1999;#Brock,#2004).#In#fact,#organza#bags#were#effective#at#preventing#pollen#movement#between#inflorescences,#but,#given#that#flower#heads#are#sessile#and#cannot#be#completely#isolated#from#the#surrounding#rosette#or#the#ground,#small#insects#incidentally#carrying#pollen#from#other#individuals#might#sometimes#be#able#to#crawl#inside#the#bags#and#reach#the#florets.#At#the#same#time,#the#isolation#treatment#did#not#hinder#the#ability#of#apomicts#to#set#seed.#Although#their#average#seed#set#was#lower#compared#to#that#of#unmanipulated#polyploids#(control),#achenes#produced#by#isolated#flower#heads#of#apomicts#were#usually#viable,#as#inferred#by#the#high#germination#rate#of#filled#achenes.#The#difference#in#seed#set#could,#at#least#partially,#have#to#do#with#the#year#in#which#the#experiment#was#completed.#The#isolation#treatment#on#apomictic#populations#was#done#in#the#spring#of#2013,#a#year#after#all#the#other#treatments.#In#the#spring#of#2013,#plants#were#exposed#to#unusually#low#temperatures#and#consecutive#snow#storms#(personal#observation),#which#likely#affected#their#reproductive#output.#These#considerations#notwithstanding,#the#outcome#of#the#isolation#treatment#confirmed#that#diploids#are#outbreeders#and#polyploids#rely#on#apomixis.#In#addition,#the#absence#of#fruit#production#observed#in#unemasculated#sexual#inflorescences#indicated#that#they#are#likely#to#be#self[incompatible,#a#trait#observed#in#sexual#relatives#of#most#other#agamic#complexes#(Asker#and#Jerling,#1992),#and#described#as#a#characteristic#of#Townsendia#(Beaman,#1957;#Tepedino#et.al.,#2004).##The#reproductive#success#of#sexual#and#apomictic#populations#of#the#Easter#Daisy#was#estimated#by#measuring#seed#set,#achene#mass,#germination#rate#and#seedling#survival#in#open[pollinated#mother#plants#(controls);#and#the#four#parameters#were#subsequently#compared#among#breeding#types.#29"Sexuals#and#apomicts#had#similar#seed#set,#as#well#as#inflorescence#of#comparable#size#based#on#the#number#of#both#filled#and#empty#achenes#produced#per#capitulum.#Apomicts#had#slightly#heavier#filled#achenes,#but#the#difference#was#not#significant.#Polyploid#achenes#had#a#lower#germination#rate#than#diploids,#however#apomictic#seedlings#had#a#higher#chance#of#surviving#and#becoming#fully#established#plants.#Overall,#the#cumulative#effects#of#breeding#system#on#the#four#different#parameters#measured#allows#me#to#conclude#that#sexual#diploids#and#apomictic#polyploids#have#similar#reproductive#success#in#the#area#where#this#study#was#carried#out,#which#represents#the#only#portion#of#the#range#where#populations#of#the#two#cytotypes#are#found#in#close#proximity.#This#means#that#apomicts#do#not#seem#to#possess#a#clear#advantage#over#their#diploid#progenitors#in#terms#of#reproductive#output,#because#they#do#not#produce#a#larger#number#nor#a#higher#proportion#of#filled#achenes#compared#to#diploid#mothers.#In#addition,#the#combined#results#of#germination#and#survival#rates,#show#that#apomictic#and#sexual#offspring#have#the#same#overall#fitness#during#the#early#stages#of#development.#Seed#dispersal#has#been#shown#to#be#important#in#the#spread#of#apomictic#forms#in#other#taxa,#such#as#Antennaria.parlinii,#where#apomicts#produce#smaller,#lighter#and#more#numerous#achenes,#with#a#consequent#increase#in#dispersal#potential#over#their#sexual#counterparts,#especially#in#disturbed#areas#(Michaels#and#Bazzaz,#1986;#O’Connell#and#Eckert,#2001).#In#T..hookeri#I#did#not#observe#any#major#difference#in#fruit#mass#or#fruit#morphology#between#diploid#and#polyploid#cytotypes.#However,#preliminary#studies#of#dispersal#traits#suggest#that#polyploids#may#be#able#to#disperse#farther#from#their#parental#plants,#because#their#fruits,#owing#to#characteristics#of#the#pappus,#have#lower#terminal#velocity#(i.e.,#they#descend#over#a#known#distance#in#still#air#at#a#slower#pace)#than#achenes#of#sexuals#(Ruiz[Larrea#and#Whitton,#unpublished).#Based#on#our#current#understanding#of#the#system,#it#appears#unlikely#that#apomicts#have#been#able#to#re[colonize#vast#portion#of#the#species#range#as#a#consequence#of#a#greater#reproductive#efficiency#resulting#from#the#reallocation#of#resources#toward#the#female#function.#30"Instead,#apomicts#might#be#more#successful#because#of#the#advantages#of#uniparental#reproduction#or#due#to#an#enhanced#dispersal#ability.#2.4.2 Experimental(crossings((SS(and(SA(treatments)(The#cross[pollination#protocol#was#successful,#based#on#the#observation#that#most#crosses#yielded#some#filled#achenes#and#it#was#possible#to#compare#the#reproductive#success#of#seed#parents#receiving#pollen#from#apomictic#(SA)#versus#sexual#(SS)#donors.#The#method#used#to#cross[pollinate#inevitably#produces#a#mixture#of#self#and#cross#pollen#on#the#stigma#of#recipient#florets,#but#the#presence#of#self[pollen#should#not#have#compromised#the#outcome#of#the#crosses,#because#diploids#are#likely#to#be#self[incompatible.##Seed#set#was#significantly#lower#in#plants#treated#with#heterospecific#(SA)#rather#than#conspecific#(SS)#pollen#in#population#2030#(2[day#pollination).#Reduced#fruit#production#in#polyploid[diploid#compared#to#diploid[diploid#crosses#has#been#reported#in#a#number#of#studies#on#Taraxacum#(Richards,#1970;#Morita#et.al.,#1990a;#Tas#and#Van#Dijk,#1999),#and#can#be#attributed#to#the#low#quality#pollen#produced#by#apomicts,#i.e.#to#the#availability#of#fewer#viable#grains#compare#to#the#pollen#produced#by#sexuals#(Thompson#and#Whitton,#2006;#Garani#et.al.,#unpublished)#or#to#the#early#abortion#of#zygotes#sired#by#aneuploid#grains#(Richards,#1997;#Tas#and#Van#Dijk,#1999).#In#population#2033#(1[day#pollination)#the#two#treatments#(SS#and#SA)#produced#similar#average#seed#set,#but#in#the#SA#treatment#the#seed#set#of#individual#seed#parents#was#more#widely#spread#around#the#mean,#with#more#than#30%#of#plants#failing#to#set#seed#entirely#and#another#24%#having#high#seed#set#(between#31%#and#50%).#It#is#unlikely#that#this#variation#was#due#to#pollen#amount#or#quality#alone,#because#each#recipient#inflorescence#was#pollinated#with#two#flower#heads#collected#from#two#distinct#pollen#donors,#but#at#this#stage#it#is#not#possible#to#determine#what#caused#the#observed#pattern.#Moreover,#in#both#populations#seed#set#of#crossed#individuals#(especially#in#31"sexual[sexual#crosses)#was#low#compared#to#that#of#open[pollinated#plants#(controls),#likely#because#of#pollen#limitation.#It#must#be#noted#that#the#florets#in#a#flower#head#are#not#receptive#simultaneously,#but#open#sequentially#inward.#Because#the#crosses#were#performed#mainly#when#the#outer#disk#florets#were#in#anthesis,#it#is#possible#that#no#viable#pollen#was#available#by#the#time#the#more#central#florets#matured.#Brock#(2004)#encountered#a#similar#issue#when#crossing#dandelions#in#the#field,#and#attributed#the#reduced#seed#set#of#crossed#compare#to#unmanipulated#plants#to#the#use#of#an#insufficient#number#of#donor#florets.#Repeating#the#treatment#over#a#longer#period#of#time#may#have#resolved#this#issue,#however#achieving#full#pollination#in#experimental#crosses#in#the#field#can#be#complex.#Pollen#from#asexual#donors#did#not#decrease#seed#set#of#parental#plants#(relative#to#sexual[sexual#crosses)#in#2033#as#observed#in#2030,#but#achenes#from#sexual[asexual#crosses#in#population#2033#were#slightly#lighter#and#had#significantly#lower#germination#rate#than#achenes#sired#by#diploid#pollen#parents.#None#of#these#differences#was#significant#in#population#2030.#Survival#rate#was#significantly#lower#for#seedlings#from#sexual[apomictic#crosses#compared#to#offspring#from#sexual[sexual#crosses,#and#this#was#true#for#both#recipient#populations.#The#factors#causing#lower#germination#rates#in#2033#and#the#decreased#survival#of#progeny#derived#from#sexual[asexual#crosses#cannot#be#entirely#determined#at#this#stage.#Similar#results#were#obtained#with#Taraxacum,#where#it#was#proposed#that#a#decreased#germination#rate#and#lower#survival#could#be#linked#to#inbreeding#depression#in#selfed#progeny#(Tas#and#Van#Dijk,#1999).#Alternatively,#inviable#offspring#could#result#from#reproductive#incompatibilities#between#individuals#of#different#ploidy#levels,#as#observed#in#interploidy#crosses#between#sexually#reproducing#groups,#where#progeny#are#often#inviable#because#of#imbalances#in#ploidy#between#different#tissues#of#the#hybrid#seeds#(i.e.,#‘triploid#block’;#Marks,#1966;#Ramsey#and#Schemske,#1998).##32"To#summarize,#this#study#shed#light#on#two#major#potential#outcomes#of#reproductive#interactions#between#sexual#diploids#and#apomictic#polyploids#in#T..hookeri,#despite#the#limitations#and#difficulties#imposed#by#working#in#the#field#rather#than#in#a#controlled#environment.#First,#the#cross[pollination#experiment#provided#evidence#that#apomicts#can#successfully#fertilize#their#diploid#progenitors,#meaning#that#reproductive#barriers#among#cytotypes#are#not#complete.#Second,#I#found#a#consistent#trend#toward#a#decrease#in#reproductive#success#of#diploid#seed#parents#when#treated#with#pollen#of#apomictic#origin#compared#to#conspecific#pollen.#If#we#consider#that#each#trait#analyzed#represents#a#distinct#stage#of#the#early#offspring#development#(from#seed#set#to#seedling#survival),#we#can#conclude#that#the#additive#effects#of#one#stage#on#the#next#provide#substantial#support#to#my#initial#hypothesis#that#asymmetric#reproductive#interference#may#play#a#role#in#preventing#the#expansion#of#diploid#populations#northward,#into#the#apomicts#range#(e.g.,#Hörandl,#2006).#2.4.3 Flow(cytometry((FCM)(After#assessing#the#reproductive#success#of#hand[crossed#sexual#plants,#I#used#flow#cytometry#to#confirm#the#ploidy#of#both#seed#and#pollen#parents,#and#to#estimate#the#ploidy#level#of#the#progeny#that#resulted#from#sexual[apomictic#crosses.#The#analysis#was#conducted#using#a#protocol#partially#modified#from#Suda#and#Trávníček#(2006)#and#Sears#(2011),#that#I#initially#developed#for#field[collected#silica[dried#leaf#tissue#(Appendix#B).#It#is#generally#recommended#to#grind#the#tissue#of#the#standard#species#together#with#each#sample,#because#the#presence#of#specific#cytosolic#compounds#may#affect#reference#and#target#species#differently#when#the#tissues#are#not#co[chopped.#This#can#result#in#the#detection#of#amplified#differences#in#florescence#intensity#that#do#not#necessarily#reflect#real#differences#in#nuclear#DNA#content#(Bennett#et.al.,#2000;#Price#et.al.,#2000;#Doležel#et.al.,#2007).#My#co[ground#test#(Appendix#33"C),#however,#showed#that#it#is#possible#to#obtain#repeatable#2C[value#estimates#even#when#T..hookeri#and#reference#leaf#tissue#are#prepared#separately.#Moreover,#a#similar#method,#based#on#co[staining#but#independently#processing#nuclei#from#standard#and#target#species,#was#successfully#used#to#assess#the#DNA#amount#in#samples#of#Crataegus#(Talent#and#Dickinson,#2005).#The#literature#also#contains#examples#of#studies#where#the#nuclei#of#internal#standard#and#species#of#interest#were#prepared#and#stained#separately#before#being#mixed#and#analyzed#(e.g.,#Johnston#et.al.,#1996;#O’Brien#et.al.,#1996).#Diploid#parental#plants#that#received#pollen#from#polyploid#donors#produced#achenes#of#varying#ploidy,#including#DNA#contents#that#are#consistent#with#euploid#and#near[euploid#(diploid,#triploid#and#tetraploid)#as#well#as#aneuploid#cytotypes.#This#finding#is#consistent#with#results#from#similar#experimental#crossings#performed#between#diploid#mothers#and#triploid#pollen#donors#in#Taraxacum,#where#diploid,#triploid#and#tetraploid#progeny#were#detected#(Tas#and#Van#Dijk,#1999;#Verduijn#et.al.,#2004b;#Mártonfiová,#2006;#Meirmans#et.al.,#2006).#Some#studies#also#reported#the#occurrence#of#aneuploid#hybrids#(Richards,#1970;#Sterk,#1987;#Morita#et.al.,#1990b).##The#number#of#individual#offspring#within#each#ploidy[category#varied#dramatically#between#the#two#seed#parent#populations#and,#notably,#putative#tetraploids#were#produced#only#by#mothers#in#2030.#The#inconsistency#could#be#due#to#differences#between#the#pollen#source#populations,#the#recipient#populations,#or#their#interaction.#Without#further#experiments#it#is#not#possible#to#make#a#definitive#conclusion,#but#it#seems#least#likely#to#be#the#result#of#differences#between#the#pollen#sources.#In#fact,#populations#2019#and#2027#are#both#triploid#and#have#similar#average#pollen#viability#and#size.#I#did#however#observe#inherent#differences#between#the#two#recipient#populations,#which#have#been#shown#to#partially#contrast#in#their#reproductive#output#(2030#produced#significantly#more#and#larger#fruits#than#2033).#Finally,#it#is#important#to#consider#that#the#results#from#the#flow#cytometric#analysis#reflect#only#the#offspring#that#germinated#and#that#were#34"large#enough#to#provide#sufficient#leaf#material#for#the#cytological#analysis.#Thus,#FCM#allowed#the#identification#of#the#presence#of#different#cytotypes,#but#did#not#allow#quantification#of#the#proportion#of#the#total#progeny#that#fell#into#each#ploidy#category.#It#is#unlikely#that#the#most#vigorous#offspring#are#a#random#sample#of#the#seeds#that#germinated.#Further#experiments#designed#to#isolate#the#effect#of#pollen#and#maternal#sources#are#needed#to#resolve#the#causes#of#differences#in#the#progeny#obtained.##Despite#these#differences,#most#mother#plants#in#both#populations#gave#rise#to#some#diploid#progeny#when#pollinated#with#heterospecific#pollen.#These#diploid#F1#offspring#can#have#one#of#two#possible#origins:#they#can#arise#from#self[pollination#or#they#can#be#true#hybrids.#Selfed#diploids#can#form#when#the#receptive#stigma#is#saturated#with#both#heterospecific#and#conspecific#pollen,#a#mixture#that#can#induce#the#breakdown#of#self[incompatibility#systems,#in#a#phenomenon#known#as#the#“mentor[effect”#(Richards,#1997).#True#hybrids#would#result#from#the#fertilization#of#a#haploid#ovule#by#a#haploid#sperm#nucleus,#and#thus#would#carry#paternal#genes,#possibly#including#genes#for#apomixis.#The#molecular#examination#of#progeny#formed#in#analogous#crosses#revealed#that#mentor[effects#are#often#responsible#for#the#generation#of#diploid#selfed#seed,#but#that#hybrid#diploids#are#also#formed#(e.g.,#Hughes#and#Richards,#1988;#Noyes,#2000).#The#proportion#of#selfed#diploid#F1#reported#by#different#authors#is#highly#variable.#For#instance,#more#than#85%#of#the#viable#offspring#obtained#in#experimental#crosses#between#diploid#and#triploid#Taraxacum#were#the#product#of#selfing#and#no#diploid#F1#hybrids#were#identified#(Morita#et.al.,#1990a;#Tas#and#Van#Dijk,#1999).#In#contrast,#a#cross#between#diploid#sexual#Erigeron.strigosus#and#triploid#apomictic#Erigeron.annuus#generated#selfed#diploids#(representing#30.8%#of#the#progeny)#as#well#as#true#diploid#hybrids#(Noyes,#2000).#An#even#lower#proportion#of#selfed#diploid#seeds#(1.3%#of#the#progeny)#resulted#when#diploid#sexual#mother#plants#of#the#Ranunculus.auricomus#complex#received#pollen#from#tetra[#and#hexaploid#apomict#relatives#(Hörandl#and#Temsch,#2009).#Because#hand[pollination#has#been#performed#by#rubbing#the#parental#flower#heads#against#each#other#without#emasculation,#35"it#is#possible#that#mentor[effects#took#place#when#seed#parents#received#pollen#from#apomictic#donors#(selfing#generally#does#not#occur#when#crosses#are#performed#between#outbreeding#diploids#using#the#same#methods;#Morita#et.al.,#1990b;#Brock,#2004).#The#present#work#did#not#include#an#in[depth#examination#of#diploid#offspring#formation.#However,#it#is#possible#to#speculate#that,#if#diploids#are#the#product#of#selfing,#then#they#will#likely#have#lower#average#vegetative#fitness#than#outcrossed#progeny#due#to#inbreeding#depression,#as#found#by#Morita#et.al.#(1990a)#and#Van#Dijk#(2007).##Approximately#half#of#the#individuals#produced#by#mother#plants#from#population#2030#were#polyploid#(20#of#the#41#samples#examined),#whereas#only#one#polyploid#(likely#a#triploid)#was#detected#among#the#37#samples#screened#from#2033.#Triploid#and#tetraploid#progeny#can#only#have#arisen#from#the#fertilization#of#a#haploid#ovule#by#diploid#and#triploid#pollen#nuclei,#respectively.#Consequently,#such#offspring#are#always#of#hybrid#origin#(assuming#unreduced#gamete#production#in#diploids#is#negligible,#which#I#infer#based#on#pollen#observations#from#diploids).#This#also#indicates#that#triploid#fathers#are#able#to#produce#partially#reduced#diploid#(n=2x)#as#well#as#unreduced#triploid#pollen#grains#(n=3x).#Given#the#high#frequency#of#tetraploid#hybrids,#microsporogenesis#in#polyploids#may#be#biased#toward#triploid#pollen#grains#(Tas#and#Van#Dijk,#1999).#Alternatively,#it#is#possible#that#a#disturbed#meiosis,#which#is#characteristic#of#taxa#with#autonomous#apomixis#(Asker#and#Jerling,#1992;#Mogie,#1992)#often#leads#to#the#formation#of#unbalanced#and#thus#inviable#reduced#or#partially#reduced#pollen,#a#process#that#would#not#affect#unreduced#triploid#grains#if#meiosis#was#bypassed#entirely.##Meiotic#irregularities#during#microsporogenesis#could#also#generate#a#fraction#of#aneuploid#pollen#that#retains#the#ability#to#function#in#crosses#with#diploid#seed#parents,#giving#rise#to#aneuploid#offspring.#Indeed,#it#seems#unlikely#that#these#progeny#resulted#from#selfing.#In#contrast#to#what#was#observed#for#polyploids,#putative#aneuploid#hybrids#were#considerably#more#frequent#in#36"population#2033,#where#five#plants#had#a#nuclear#DNA#content#slightly#smaller#than#diploids#and#thirteen#had#genomes#intermediate#between#diploids#and#triploids.#Only#five#offspring#from#2030#fell#in#the#latter#category#and#none#in#the#former.#The#underlying#determinants#of#this#variation#are#unknown,#but#they#might#be#associated#with#altered#chromosome#numbers,#i.e.#the#result#of#the#irregular#chromosome#complement#of#the#pollen#of#triploid#parents#(Richards,#1970).#Variation#in#chromosomal#copy#number#has#been#reported#in#experimental#set[ups#analogous#to#the#present#work,#where#numerical#aneuploidy#has#been#identified#using#standard#chromosome#counts.#For#instance,#the#130#F1#Erigeron#hybrids#obtained#by#fertilizing#diploid#sexuals#(2n=2x=18)#with#pollen#from#apomictic#triploids#(2n=3x=27)#formed#a#cytologically#complex#population#that#displayed#a#bimodal#distribution#of#chromosome#numbers,#with#peaks#at#2n=18[19#and#2n=25[26#and#ranging#from#diploid#to#tetraploid#(2n=36)#(Noyes,#2000).#Similarly,#in#a#cytological#analysis#of#48#Tripsacum.dactyloides#hybrids,#obtained#from#crossing#diploid#sexual#plants#(2n=2x=36)#with#individuals#from#an#apomictic#triploid#accession#(2n=3x=54),#the#offspring#were#found#to#have#variable#chromosome#numbers#spanning#nearly#the#entire#range#from#diploid#to#triploid#(Sherman#et.al.,#1991).#Recently,#genomic#and#fluorescence#in#situ#hybridization#techniques#uncovered#extensive#and#repeated#patterns#of#chromosomal#variation#in#naturally#occurring#populations#of#the#neoallopolyploid#Tragopogon.miscellus#(Chester#et.al.,#2012).#At#this#stage,#it#is#not#possible#to#determine#exactly#how#these#putative#euploid#and#aneuploid#F1#generations#formed;#and#assessing#their#breeding#behavior#was#equally#impracticable#because,#for#reasons#that#are#still#unclear,#T.#hookeri#fails#to#flower#when#kept#in#a#growth#chamber.#Based#on#previous#work#(Richards,#1970;#Morita#et.al.,#1990a;#Morita#et.al.,#1990b),#the#reproductive#mode#of#sexual[apomictic#hybrids#tends#to#be#predicted#by#ploidy#level,#i.e.#with#diploid#hybrids#being#sexual#outcrossers#and#polyploids#being#apomictic#;#alternatively,#plants#could#be#sterile.#It#is#highly#improbable#that#diploid#offspring#would#reproduce#asexually#given#that#gametophytic#apomixis#is#strongly#correlated#with#polyploidy#and#apomictic#diploids#are#extremely#rare#in#nature#(Nogler,#37"1984;#Schranz#et.al.,#2006).#In#accordance#with#these#predictions,#Tas#and#Van#Dijk#(1999)#reported#that#one[third#of#the#triploid#and#all#of#the#tetraploid#hybrids#reproduced#via#apomixis,#whereas#none#of#their#four#diploid#hybrids#was#apomictic.##Given#these#results#it#is#possible#to#conclude#that#apomictic#individuals#have#the#potential#to#reduce#the#reproductive#success#of#their#sexual#progenitors#in#experimental#crosses.#In#fact,#the#product#of#this#unidirectional#interaction#was#an#array#of#diploid,#polyploid#and#aneuploid#descendants#that#might#have#reduced#fitness#or#might#be#sterile#as#a#result,#for#instance,#of#inbreeding#depression#(selfed#diploids)#or#chromosomal#instability.#It#is#also#reasonable#to#expect#that#at#least#a#portion#of#such#offspring#is#fully#apomictic#or#possesses#apomixis#genes#and#will#pass#these#traits#to#their#progeny.#It#should#be#noted#that#while#apomictic#hybrid#offspring#would#contribute#to#the#reproductive#fitness#of#their#diploid#maternal#plants,#in#nature,#these#apomicts#could#shift#the#relative#frequencies#of#the#two#mating#types.#In#conclusion,#these#findings#contribute#towards#explaining#why#apomictic#and#sexual#forms#have#not#been#observed#to#co[occur#in#this#taxon#and#why#outcrossing#diploids#have#not#been#found#in#the#central#and#northern#portion#of#the#range#(outside#Yukon).#In#fact,#this#study#showed#that#the#persistence#of#sexual#cytotypes#could#be#imperiled#in#the#presence#of#a#large#number#of#apomictic#neighbours,#while#being#surrounded#by#sexuals#will#have#no#impact#on#apomictic#individuals,#given#that#they#do#not#require#pollen#for#embryo#or#endosperm#formation#and#development.#As#a#result,#apomicts#might#have#the#ability#to#wipe#out#co[occurring#diploid#individuals#at#local#scale,#creating#a#barrier#that#prevents#sexuals#from#spreading#into#territories#dominated#by#asexual#lineages.#Thus,#the#presence#of#diploid#sexual#populations#could#be#restricted#only#to#areas#of#the#range#where#they#hold#a#selective#advantage#over#polyploids#and#represent#the#large#majority#of#individuals#(Mogie,#1992;#Mogie#et.al.,#2007;#Whitton#et.al.,#2008).##  38!Table&2.1!Identification!numbers,!site!descriptions!(with!GPS!coordinates!and!altitude),!pollen!data!(stainability,!average!size!and!standard!deviation!of!viable!grains)!and!ploidy!estimates!for!the!populations!of!Townsendia*hookeri!selected!to!conduct!the!cross?pollination!experiment,!including!population!2031!(described!in!chapter!3).!Pollen!stainability!and!size!were!measured!following!the!methodology!described!in!Appendix!A.!ID& Locality& Latitude&/&Longitude&(decimal)& Alt.&(m)& Mean&pollen&stainability&(%)& Mean&pollen&diameter&(µm)& Std.&Dev.&(µm)& Estimated&ploidy&2030! 19!km!E!of!Laramie!(WY),!Medicine!Bow!National!Forest!?!exit!#323!(Lincoln!Memorial!rest!area)!on!Hwy!80!41.24002!?105.43423! 2663! 94.13! 22.14! 0.96! Diploid!2032! 26!km!E!of!Laramie!(WY),!Medicine!Bow!National!Forest!?!exit!#329!(Vedauwoo!Glen!Rd);!drive!~!3!km!E!along!Vedauwoo!Glen!Rd.;!plants!are!found!on!both!side!of!the!road.!41.15272!?105.36605! 2510! 91.6! 21.73! 1.28! Diploid!2033! 26!km!E!of!Laramie!(WY),!Medicine!Bow!National!Forest!?!exit!#329!(Vedauwoo!Glen!Rd);!drive!~!500!m!along!US!Forest!Service!Rd!700!G!from!junction!with!Vedauwoo!Glen!Rd.!(second!dirt!road!from!the!exit,!on!the!NW!side!of!the!road);!on!W!side!of!the!road.!41.16206!?105.40258! 2553! 93.65! 22.15! 1.25! Diploid!2035! 30!km!E!of!Laramie!(WY),!Medicine!Bow!National!Forest!?!exit!#329!(Vedauwoo!Glen!Rd);!drive!~!4!km!E!along!Vedauwoo!Glen!Rd.;!on!W!side!of!the!road.!41.15334!?105.35812! 2532! 92.36! 22.02! 1.02! Diploid!39!ID& Locality& Latitude&/&Longitude&(decimal)& Alt.&(m)& Mean&pollen&stainability&(%)& Mean&pollen&diameter&(µm)& Std.&Dev.&(µm)& Estimated&ploidy&2019! E!Laramie!(WY)!?!2!km!NW!along!Old!Stone!Quarry!Rd!(dirt!road)!from!junction!with!E!Grand!Ave/Hwy!80.!The!dirt!road!is!800!m!W!from!the!intersection!of!E!Gran!Ave!and!Boulder!Dr.!41.30645!?105.52158! 2313! 33.75! 33.02! 3.24! Polyploid!2027! 40!km!W!of!Laramie!(WY),!Medicine!Bow!National!Forest!?!drive!!3.6!km!N!along!Fox!Creek!Rd/Co!Rd!47!from!junction!with!230.!From!parking!spot!on!S!side!of!the!road!(next!to!river)!walk!~!300!m!N!along!a!dry!creek,!then!go!through!the!U.S.!Forest!Service!fence!on!the!right.!41.14187!?106.04396! 2433! 28.19! 33.47! 4.2! Polyploid!!2031! 17!km!NE!of!Laramie!(WY)!on!Roger!Canyon!Road!?!16.6!road!km!from!junction!with!Grand!Avenue;!13.8km!from!point!where!Roger!Canyon!changes!from!9th!Street..!41.3864!?105.47349! 2359! 25.53! 33.75! 5.45! Polyploid!40!Table&2.2!Summary!of!the!work!completed!in!the!field!during!the!spring!of!2012!and!2013.!For!each!population,!the!table!reports:!the!identification!number!(ID),!function!(SR!=!sexual!recipient,!SD!=!sexual!donor,!AD!=!apomictic!donor),!replicate!experiment!(R1!or!R2),!treatment(s)!done,!year!when!the!treatment(s)!was!conducted!and!number!of!days!over!which!the!pollination!was!repeated.!ID& Function& Replicate& Treatment& Year& Days&of&pollination&2030! SR! R1! C,!I,!SS,!SA! 2012! 2!2032! SD! R1! JJ! JJ! JJ!2033! SR! R2! C,!I,!SS,!SA! 2012! 1!2035! SD! R2! JJ! JJ! JJ!2019! AD! R1! C,!I! 2012/2013! JJ!2027! AD! R2! C! 2012! JJ!2031! JJ! JJ! I! 2013! JJ!Table&2.3!Summary!of!twoJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!treatment!(control!vs.!isolation),!reproduction!(sexual!vs.!apomictic!breeding!system)!and!the!interaction!term!on!seed!set.!The!analysis!was!done!using!GLM!with!family!=!quasibinomial!to!account!for!overdispersion!(meaning!that!the!variance!of!the!error!distribution!in!the!data!was!greater!than!that!expected!under!the!binomial!distribution).!The!significance!of!terms!was!assessed!with!an!FJtest.&Bold!values!are!significant!at!p<0.05.!!Seed!set! SS! d.f.! F! Pr(>F)!Treatment! 600.98! 1! 181.14! <2.20e?16&Reproduction! 425.53! 1! 128.26! <2.20e?16&Treatment!:!Reproduction! 619.63! 1! 186.77! <2.20e?16&Residuals! 242.19! 73! ! !& &41!Table&2.4!Summary!of!oneJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!reproduction!(sexual!vs.!apomictic!breeding!system)!on!germination!rate.!The!analysis!was!done!using!GLM!with!family!=!quasibinomial.!The!significance!of!terms!was!assessed!with!an!FJtest.!Bold!value!is!significant!at!p<0.05.!Germination! SS! d.f.! F! Pr(>F)!Reproduction! 12.66! 1! 4.4417! 0.0421&Residuals! 102.61! 36! ! !Table&2.5!Summary!of!oneJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!reproduction!(sexual!vs.!apomictic!breeding!system)!on!seedling!survival!rate.!The!analysis!was!done!using!GLM!with!family!=!binomial.!The!significance!of!terms!was!assessed!with!a!ChiJsquare!test.!Bold!value!is!significant!at!p<0.05.!Survival! d.f.! Deviance! Pr(>F)!Reproduction! 1! 4.1017! 0.04284&Residuals! 36! 46.624! !Table&2.6!Summary!of!oneJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!treatment!(sexualJsexual!crosses!vs.!sexualJapomictic!crosses)!on!seed!set!for!the!two!pollen!recipient!populations.!The!analysis!was!done!using!GLM!with!family!=!quasibinomial.!The!significance!of!terms!was!assessed!with!an!FJtest.!Bold!value!is!significant!at!p<0.05.!Population*2030*Seed!set! SS! d.f.! F! Pr(>F)!Treatment! 66.05! 1! 9.0154! 0.004279&Residuals! 344.34! 47! ! !Population*2033*Seed!set! SS! d.f.! F! Pr(>F)!Treatment! 3.16! 1! 0.2814! 0.5982&Residuals! 539.81! 48! ! !42!Table&2.7!Summary!of!oneJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!treatment!(sexualJsexual!crosses!vs.!sexualJapomictic!crosses)!on!germination!rate!for!the!two!pollen!recipient!populations.!The!analysis!was!done!using!GLM!with!family!=!binomial.!The!significance!of!terms!was!assessed!with!a!ChiJsquare!test.!Bold!value!is!significant!at!p<0.05.!Population*2030*Germination! d.f.! Deviance! Pr(>F)!Treatment! 1! 0.2467! 0.6194!Residuals! 46! 86.384! !Population*2033*Germination! d.f.! Deviance! Pr(>F)!Treatment! 1! 5.926! 0.01492&Residuals! 38! 97.149! !Table&2.8!Summary!of!oneJway!factorial!Analysis!of!Deviance!testing!for!the!effects!of!treatment!(sexualJsexual!crosses!vs.!sexualJapomictic!crosses)!on!seedling!survival!rate!for!the!two!pollen!recipient!populations.!The!analysis!was!done!using!GLM!with!family!=!binomial.!The!significance!of!terms!was!assessed!with!a!ChiJsquare!test.!Bold!values!are!significant!at!p<0.05.!Population*2030*Survival! d.f.! Deviance! Pr(>F)!Treatment! 1! 4.4422! 0.03506&Residuals! 28! 51.191! !Population*2033*Survival! d.f.! Deviance! Pr(>F)!Treatment! 1! 6.1709! 0.01299&Residuals! 21! 57.635! ! 43!!Figure'2.1'Locations)of)the)six)populations)of)Townsendia*hookeri)used)to)complete)the)cross4pollination)experiment.)Apomictic)populations)(IDs:)2019)and)2027))are)represented)with)blue)circles)and)sexual)populations)(IDs:)2030,)2032,)2033)and)2035))are)represented)with)red)circles.)The)figure)also)shows)the)location)of)the)apomictic)population)2031)(described)in)section)2.2.2).)44!)Figure'2.2'One)replicate)block)of)experimental)treatments)completed)in)the)field)on)three)natural)populations)of)Townsendia*hookeri.)Treatments)are)indicated)with)capital)letters:)C,)control;)I,)isolation;)SS,)sexual4sexual)cross;)SA,)sexual4apomictic)cross.)The)number)of)individuals)exposed)to)each)treatment)is)indicated)in)parenthesis.)Plants)are)represented)as)having)a)single)inflorescence)to)indicate)that)only)one)flower)head)per)individual)was)used)in)a)given)treatment.)Blue)inflorescences)represent)apomictic)plants)while)red)inflorescences)represent)sexual)plants.).SS!!(x25)!SA!!(x25)!I$(x10)$$C$(x10)$$I$(x10)$$C$(x10)$$Apomictic!Donor!(AD)!(!≥60!plants)!Sexual!Recipient!(SR)!(≥70!plants)!Sexual!Donor!(SD)!(≥50!plants)!45!!Figure'2.3'Achenes!of!Townsendia*hookeri!were!classified!as!“filled”!or!“empty”!based!on!the!following!criteria:!“filled”!achenes!were!plump!and!dark!brown!(first!and!second!achenes!from!the!left),!whereas!“empty”!achenes!were!flat!and!lacked!pigmentation!(fifth!and!sixth!achenes!from!the!left).!Light!brown!achenes!were!scored!as!“filled”!when!fleshy!(third!achene!from!the!left)!and!as!“empty”!when!clearly!devoid!of!an!embryo!(fourth!achene!from!the!left).!46!!Figure'2.4'Example!of!fully!established!individual!of!Townsendia*hookeri,!characterized!by!a!large!root!system!and!five!true!leaves.!1 cm 47! Figure'2.5'Cytogram!of!the!transit!time!of!propidium!iodideEstained!nuclei!through!the!laser!interrogation!point!(FL2EW)!versus!their!total!fluorescence!(FL2EA),!obtained!via!flow!cytometry.!This!type!of!plot!was!created!using!FlowJo!vX.0.6!and!it!was!used!to!identify!G0/G1!populations!of!nuclei!belonging!to!the!internal!standard!(Vicia*faba*cv.!Inovec!in!this!example)!and!the!target!tissue!(polyploid!Townsendia*hookeri).!The!two!subpopulations!of!nuclei!(in!light!green)!were!isolated!from!the!debris!background!(in!blue)!using!the!Autogate!function.!FL2-W FL2-A V. faba T. hookeri 48! Figure'2.6'Total!number!of!filled!and!empty!achenes!per!seed!head!(capitulum).!Each!head!(indicated!by!a!circle)!represents!an!apomictic!(blue)!or!sexual!(red)!seed!parental!plant!under!control!or!isolation!treatment.!The!black!circles!and!bars!are!means!and!standard!errors.!Different!shades!of!color!define!single!populations:!2019!is!dark!blue,!2027!and!2031!are!light!blue,!2030!is!dark!red!and!2033!is!light!red.!' !Total number of achenes/capitulum0255075100125150175ApomicticControl(n=18)SexualControl(n=20)ApomicticIsolation(n=20)SexualIsolation(n=19)●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●49! Figure'2.7'Percentage!of!filled!achenes!per!seed!head!(seed!set).!Each!head!(indicated!by!a!circle)!represents!an!apomictic!(blue)!or!sexual!(red)!seed!parental!plant!under!control!or!isolation!treatment.!The!black!circles!and!bars!are!means!and!standard!errors.!The!asterisk!(*)!represents!significantly!different!values!(p<0.05)!based!on!the!Analysis!of!Deviance.!Different!shades!of!color!define!single!populations:!2019!is!dark!blue,!2027!and!2031!are!light!blue,!2030!is!dark!red!and!2033!is!light!red.!    Seed set (%)01020304050607080ApomicticControl(n=18)SexualControl(n=20)ApomicticIsolation(n=20)SexualIsolation(n=19)●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●● ●●●●●●●●●●●●●●●●*  50  Figure'2.8'Percentage)germination)rate)per)filled)achene.)Each)circle)represents)the)mean)value)for)an)apomictic)(blue))or)sexual)(red))seed)parental)plant)under)control)treatment.)The)black)circles)and)bars)are)means)and)standard)errors.)The)asterisk)(*))represents)significantly)different)values)(p<0.05))based)on)the)Analysis)of)Deviance.)Different)shades)of)color)define)single)populations:)2019)is)dark)blue,)2027)is)light)blue,)2030)is)dark)red)and)2033)is)light)red.) Figure'2.9'Percentage)survival)rate)per)seedling)transplanted)from)agar)to)soil.)Each)circle)represents)the)mean)value)for)an)apomictic)(blue))or)sexual)(red))seed)parental)plant)under)control)treatment.)The)black)circles)and)bars)are)means)and)standard)errors.)The)asterisk)(*))represents)significantly)different)values)(p<0.05))based)on)the)Analysis)of)Deviance.)Different)shades)of)color)define)single)populations:)2019)is)dark)blue,)2027)is)light)blue,)2030)is)dark)red)and)2033)is)light)red.)Germination rate per filled achene (%)20406080100ApomicticControl(n=18)SexualControl(n=20)●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●Survival rate per transplanted seedling (%)020406080100ApomicticControl(n=18)SexualControl(n=20)●●●●●●●●●●●●●●●●●●●●●●●●●●●●●* * 51! Figure'2.10'Percentage)of)filled)achenes)per)seed)head)(seed)set).)Each)head)(indicated)by)a)circle))represents)a)seed)parental)plant)under)sexualKsexual)(SS))or)sexualKapomictic)(SA))crossing)treatment.)Dark)grey)circles)correspond)to)population)2030,)while)light)grey)circles)correspond)to)population)2033.)The)red)circles)and)bars)are)means)and)standard)errors.)The)asterisk)(*))represents)significantly)different)values)(p<0.05))based)on)the)Analysis)of)Deviance)done)between)treatments)within)each)population.)  TreatmentSeed set (%)01020304050SS(n=25)SA(n=24)SS(n=25)SA(n=25)●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●* 52! Figure'2.11'Average)mass)(in)mg))of)filled)achenes)per)seed)head.)Each)head)(indicated)by)a)circle))represents)a)seed)parental)plant)under)sexual:sexual)(SS))or)sexual:apomictic)(SA))crossing)treatment.)Dark)grey)circles)correspond)to)population)2030,)while)light)grey)circles)correspond)to)population)2033.)The)red)circles)and)bars)are)means)and)standard)errors.)Differences)between)treatments)within)each)population)are)not)significant)based)on)the)T:test)(2030:)t=:0.3347,)df=46,)P=0.7393;)2033:)t=1.7632,)df=37,)P=0.0861).) TreatmentAverage filled achene mass (mg)0.40.60.81.01.2SS(n=25)SA(n=23)SS(n=23)SA(n=16)●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●53! Figure'2.12'Percentage)germination)rate)per)filled)achene.)Each)circle)represents)the)mean)value)for)a)seed)parental)plant)under)sexual:sexual)(SS))or)sexual:apomictic)(SA))crossing)treatment.)Dark)grey)circles)correspond)to)population)2030,)while)light)grey)circles)correspond)to)population)2033.)The)red)circles)and)bars)are)means)and)standard)errors.)The)asterisk)(*))represents)significantly)different)values)(p<0.05))based)on)the)Analysis)of)Deviance)done)between)treatments)within)each)population.) TreatmentGermination rate per filled achene (%)020406080100SS(n=25)SA(n=23)SS(n=23)SA(n=17)●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ● ●●* 54! Figure'2.13'Percentage)survival)rate)per)seedling)transplanted)from)agar)to)soil.)Each)circle)represents)the)mean)value)for)a)seed)parental)plant)under)sexual:sexual)(SS))or)sexual:apomictic)(SA))crossing)treatment.)Dark)grey)circles)correspond)to)population)2030,)while)light)grey)circles)correspond)to)population)2033.)The)red)circles)and)bars)are)means)and)standard)errors.)The)asterisks)(*))represent)significantly)different)values)(p<0.05))based)on)the)Analysis)of)Deviance)done)between)treatments)within)each)population.)' 'TreatmentSurvival rate per transplanted seedling (%)020406080100SS(n=8)SA(n=22)SS(n=8)SA(n=15)●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●● ●●* * 55!!!     Figure'2.14'Genome!size!(2C-value)!of!plants!from!seed!or!pollen!parental!populations!(including!2031),!calculated!performing!flow!cytometry!on!silica-dried!leaf!tissue.!The!number!of!samples!analyzed!for!each!population!(n)!is!reported!in!the!legend.!Dotted!lines!delimit!size!ranges!associated!with!diploid!(2C-v.!<!14!pg;!in!red)!and!triploid!(17!pg!<!2C-v.!<!21!pg;!in!blue)!genomes.!Data!points!are!spread!along!the!y-axis!to!better!visualize!overlapping!values.! Figure'2.15'Genome!size!(2C-value)!of!offspring!from!sexual-apomictic!crosses!in!populations!2030!(n=41)!and!2033!(n=37),!calculated!performing!flow!cytometry!on!silica-dried!leaf!tissue.!Dotted!lines!delimit!size!ranges!associated!with!diploid!(11!pg!<!2C-v.!<!14!pg;!in!red)!and!polyploid!(2C-v.!>!17!pg;!in!blue)!genomes.!Orange!and!brown!circles!represent!nuclear!DNA!contents!consistent!with!an!aneuploid!condition.!Data!points!are!spread!along!the!y-axis!to!better!visualize!overlapping!values.! !2C−value (pg)9 11 13 15 17 19 21 23 25 27●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●● ●●●●●2033●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●● ●●20302C−value (pg)12 13 14 15 16 17 18 19 20 21●●●●●●●●●● ●●●●● ●●●●●●●●●●Population2030 (n=7)2033 (n=5)2019 (n=4)2027 (n=8)2031 (n=2)●●●●●56!Chapter(3: Conclusions(3.1 General(conclusions((This%research%explored%possible%factors%that%have%contributed%to%and%maintain%the%current%geographical%distribution%of%sexual%diploid%and%apomictic%polyploid%populations%of%the%Easter%Daisy%across%the%species’%range.%The%study%was%conducted%on%populations%located%in%the%southern%portion%of%the%range%(southern%Wyoming),%where%sexual%and%asexual%populations%are%found%in%close%proximity,%and%thus%where%contact%between%breeding%types%is%more%likely%to%have%occurred%in%the%past%or%could%occur%in%the%future.%Working%in%the%field%had%the%benefit%that%plants%were%maintained%in%their%natural%habitat,%but%it%also%imposed%some%limitations%owing%to%adverse%weather%conditions%(mostly%snow%covering%the%plants)%and%other%unpredictable%natural%events.%Consistent%with%Beaman’s%results%(1957),%the%isolation%treatment%indicated%that%diploids%do%require%mating%partners%for%successful%seed%set,%whereas%polyploids%are%apomictic%and%able%to%sire%viable%progeny%in%the%absence%of%external%pollen.%Diploid%and%polyploid%cytotypes%were%found%to%have%similar%reproductive%success%under%open%pollination,%suggesting%that%apomixis%does%not%represent%a%clear%advantage%in%terms%of%number%or%size%of%achenes%produced%under%natural%conditions,%at%least%for%these%populations%during%the%year%of%observation.%Lastly,%this%work%confirmed%the%prediction%that%apomicts%retain%the%ability%to%fertilize%their%sexual%progenitors%through%their%pollen,%and%that%this%unidirectional%interaction%is%disadvantageous%for%diploids.%When%sexual%seed%parents%received%heterospecific%rather%than%conspecific%pollen,%seed%set%decreased,%the%germination%rate%of%achenes%was%lower%and%seedlings%were%less%likely%to%survive%and%become%fully%established%plants.%In%addition,%the%fraction%of%the%progeny%that%survived%had%variable%ploidy%levels,%consistent%with%diploid,%triploid%and%tetraploid,%but%also%aneuploid%cytotypes.%The%breeding%system%of%F1%hybrids%was%not%tested,%but%it%is%expected%that%at%least%some%polyploid%offspring%would%be%fully%apomictic%(Richards,%1970;%Morita%et#al.,%1990b)%or%at%least%possess%apomixis%genes%(e.g.,%Noyes,%2006).%Based%on%these%lines%of%57!evidence,%reproductive%interference%might%contribute%to%explaining%why%sexual%forms%of%Townsendia#hookeri%are%restricted%to%the%unglaciated,%southernmost%part%of%the%range,%and%failed%to%spread%northward,%into%the%territory%occupied%by%their%apomictic%descendants.%In%a%postglacial%reTcolonization%scenario,%it%is%possible%that%faster%moving%apomictic%lineages%have%established%asexual%populations%in%newly%opened%portions%of%the%range,%and%that%subsequently%these%populations%have%become%impenetrable%to%invasion%by%slowerTmoving%sexual%progenitors%thanks%to%the%detrimental%effects%of%their%pollen%on%the%reproductive%success%of%lowTdensity%coToccurring%sexual%types%(Mogie,%1992;%Mogie%et#al.,%2007;%Whitton%et#al.,%2008).%%3.2 Implications((The%occurrence%of%apomictically%reproducing%plants%characterized%by%a%distinct%and%widespread%geographical%distribution%make%the%Easter%Daisy%an%ideal%system%to%investigate%the%coexistence%of%apomixis%and%sexuality%within%a%taxon,%as%well%as%to%ask%questions%about%the%factors%that%determine%geographical%parthenogenesis,%a%pattern%described%in%a%number%of%plant%groups%but%still%not%well%understood%(reviewed%by%Hörandl,%2006).%Although%most%likely%only%about%1%%of%flowering%plant%species%are%apomictic%(Whitton%et#al.,%2008),%including%representatives%in%28%orders%and%33%families%(Carman,%1997),%apomixis%represents%a%fundamental%alternative%strategy%to%the%predominant%sexual%pathways.%In%recent%years%the%study%of%this%peculiar%reproductive%strategy%has%gathered%much%attention,%especially%for%its%potential%application%in%crop%production.%According%to%Vielle%Calzada%et#al.,%“If%the%process%of%apomixis%can%be%understood%and%harnessed,%the%benefits%for%agriculture%are%potentially%enormous”%(Vielle%Calzada%et#al.,%1996).%Apomixis%allows%the%perpetuation%of%a%fixed%genotype%through%generations%by%avoiding%meiosis%and%ovule%fertilization%by%male%gametes%(Grimanelli%et#al.,%2001).%Thus,%this%trait%could%make%it%possible%to%perpetuate%hybrid%cultivars%and%other%varieties%with%desirable%gene%combinations%via%58!clonal%seeds,%independently%of%pollination%and%fertilization%(Koltunow%et#al.,%1995;%Spillane%et#al.,%2001).%Despite%the%great%interest%and%the%even%larger%number%of%related%scientific%publications,%little%is%known%about%how%apomictic%crops%might%interact%with%their%wild%relatives.%It%has%been%recognized%that%domesticated%plant%species%can%hybridize%with%their%wild%progenitors%(Ellstrand,%1992;%Ellstrand%et#al.,%1999).%It%is%also%known%that%most%apomicts%are%able%to%produce%some%functional%pollen,%because%apomixis%affects%the%female%reproductive%pathway,%but%has%no%direct%impacts%on%microsporogenesis%(e.g.,%Nogler,%1984).%As%a%result,%it%is%reasonable%to%expect%that%pollenTmediated%gene%flow%could%take%place%and%possibly%have%detrimental%effects%on%wild%relatives%found%within%mating%distance%of%the%newly%introduced%apomictic%crop%varieties.%Research%conducted%on%natural%plant%populations%suggested%that%when%nonTindigenous%apomictic%species%are%introduced%to%a%new%area,%they%can%threaten%the%persistence%of%native%sexual%relatives%via%genetic%assimilation%(Brock,%2004)%or%other%forms%of%reproductive%interference%(Matsumoto%et#al.,%2010).%Similarly,%experimental%studies%(e.g.,%Morita%et#al.,%1990b;%Tas%and%Van%Dijk,%1999;%Noyes,%2000)%and%the%work%presented%here%showed%how%the%male%function%of%apomictic%plants%can%interfere%with%the%normal%sexual%reproduction%of%diploid%related%forms.%Therefore,%the%‘harnessing’%of%apomixis%and%its%application%in%agriculture%will%definitely%require%a%greater%understanding%of%the%ecological%implication%of%this%trait%(such%as%gene%flow%patterns%mediated%by%male%gametes)%to%properly%predict%the%impact%that%novel%crop%varieties%will%have%on%natural%systems.%%A%better%understanding%of%the%dynamics%that%regulate%the%coexistence%of%sexual%and%apomictic%breeding%types%(i.e.,%geographical%parthenogenesis)%would%also%promote%the%implementation%of%more%effective%conservation%actions.%Diploid%cytotypes%play%a%crucial%evolutionary%role%in%an%agamic%complex,%because:%(1)%they%are%the%only%sexually%reproducing%individuals%(thus,%they%harbor%the%potential%to%generate%genetic%diversity),%and%(2)%they%give%rise%to%all%polyploid%lineages%(Babcock%and%Stebbins,%1938).%Therefore,%the%persistence%of%sexual%diploids%is%critical%for%the%longTterm%survival%of%the%species,%including%sexual%and%asexual%reproductive%types.%At%the%same%time,%in%T.#hookeri%and%59!other%similar%taxa,%diploids%are%much%less%common%than%their%apomictic%descendants,%and%their%distribution%is%restricted%within%the%overall%species’%range.%Given%that%diploid%sexuals%are%both%irreplaceable%and%uncommon,%their%populations%should%receive%special%attention%when%evaluating%the%status%of%the%species%across%its%range,%and%when%planning%management%or%conservation%programs.%3.3 Future(directions(While%this%research%has%highlighted%the%disruptive%effects%that%apomictic%polyploids%can%exert%on%the%reproductive%success%of%sexual%progenitors%through%pollen,%the%next%steps%are%to%confirm%hybridity,%assess%fitness%and%further%characterize%the%reproductive%strategy%of%the%progeny%from%sexualTapomictic%crosses%(ideally,%by%inducing%flower%development%and%assessing%seed%set%performing%the%control%and%isolation%treatments%described%in%this%study).%Reproductive%interference%is%likely%to%be%only%one%of%the%factors%that%has%influenced%the%spread%of%sexual%and%asexual%breeding%strategies%in%the%Easter%Daisy,%and%further%hypotheses%await%testing.%For%instance,%it%has%been%suggested%that%abiotic%factors%could%have%influenced%the%geographic%distribution%of%sexualTasexual%species%complexes,%both%at%large%and%small%scale%(Verduijn%et#al.,%2004a).%It%follows%that%the%absence%of%apomictic%polyploids%within%the%habitat%dominated%by%diploids%could%result%from%the%existence%of%divergent%edaphic%tolerances%between%breeding%types%that%confer%a%selective%advantage%to%sexual%over%asexual%populations%in%the%presence%of%specific%soil%conditions.%To%test%this%hypothesis,%Garani%et#al.%(unpublished)%collected%soil%samples%from%25%populations%of%T.#hookeri%scattered%throughout%most%of%the%species’%range,%from%Colorado%to%southern%British%Columbia.%The%soil%samples%were%analyzed%for%their%chemical%and%physical%properties,%but%a%careful%examination%of%the%data%has%not%yet%been%completed.%A%future%analysis%and%interpretation%of%this%large%data%set,%coupled%with%results%from%ecological%niche%modeling%(Lee%and%Whitton,%unpublished),%60!will$shed$light$on$the$contribution$of$abiotic$factors$in$shaping$the$observed$geographical$distribution$of$T.#hookeri$cytotypes$from$Colorado$to$southern$Canada.$$In$an$attempt$to$test$hypotheses$of$ecological$divergence$of$cytotypes,$PhD$student$E.$Hersh$is$setting$up$a$large$common$garden$experiment$that$includes$eight$transplant$gardens$in$three$regions$within$the$species’$native$range.$The$gardens$will$provide$a$unique$opportunity$to$investigate$the$existence$of$differences$in$fitnessCrelated$traits$(such$as$survival$and$reproductive$ability)$between$sexual$and$apomictic$plants,$and$also$to$further$test$the$reproductive$interference$hypothesis.$Thus,$this$ambitious$project$promises$to$fill$some$of$the$major$gaps$in$our$knowledge$of$the$circumstances$that$contributed$to$the$great$success$of$asexual$lineages$in$Townsendia#hookeri,$but$also$in$other$apomictic$complexes$within$and$outside$the$genus.$$ 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 R)Core)Development)Team)(2014).)R)version)2.15.0.)R)Foundation)for)Statistical)Computing,)Vienna,)Austria.)Radford)AE,)Dickison)WC,)Massey)JR,)Bell)CR)(1974).)Vascular)plant)systematics.)Harper)and)Row:)New)York.)Ramsey)J,)Schemske)DW)(1998).)Pathways,)mechanisms,)and)rates)of)polyploid)formation)in)flowering)Plants.)Annual)Review)of)Ecology)and)Systematics)29:)467–501.)Richards)AJ)(1970).)Hybridization)in)Taraxacum.)New)Phytologist)69:)1103)–)1120.)Richards)AJ)(1997).)Plant)breeding)systems,)2nd)edn.)Chapman)and)Hall:)London.)Sarkar)D)(2008).)Lattice:)multivariate)data)visualization)with)R.)Springer:)New)York.)Schranz)ME,)Kantama)L,)de)Jong)H,)MitchellHOlds)T)(2006).)Asexual)reproduction)in)a)close)relative)of)Arabidopsis:)a)genetic)investigation)of)apomixis)in)Boechera)(Brassicaceae).)The)New)Phytologist)171:)425–438.)Sears)CJ)(2011).)Systematic)investigations)into)the)North)American)Crepis)agamic)complex.)PhD)dissertation.)University)of)British)Columbia,)Vancouver.)Sherman)RA,)Voigt)PW,)Burson)BL,)Dewald)CL)(1991).)Apomixis)in)diploid)×)triploid)Tripsacum)dactyloides)hybrids.)Genome)34:)528–532.)Spillane)C,)Steimer)A,)Grossniklaus)U)(2001).)Apomixis)in)agriculture:)the)quest)for)clonal)seeds.)Sexual)Plant)Reproduction)14:)179–187.)Stanley)RG,)Linskens)HF)(1974).)Pollen)biology)biochemistry)management.)SpringerHV.)Springer)Berlin)Heidelberg:)New)York.)Stebbins)GL)(1950).)Variation)and)evolution)in)plants.)Columbia)University)Press:)New)York.)Sterk)AA)(1987).)Aspects)of)the)population)biology)of)sexual)dandelions)in)the)Netherlands.)In:)Huiskes)AHL,)Blom)CWPM,)Rozema)J)(eds).)Vegetation)between)land)and)sea,)Junk:)Dordrecht,)Netherlands.)pp)284–291.)Suda)J,)Trávníček)P)(2006).)Estimation)of)relative)nuclear)DNA)content)in)dehydrated)plant)tissues)by)flow)cytometry.)Current)Protocols)in)Cytometry)(Suppl.(38):)7.30.1–7.30.14.)Takakura)KHI,)Nishida)T,)Matsumoto)T,)Nishida)S)(2009).)Alien)dandelion)reduces)the)seedHset)of)a)native)congener)through)frequencyHdependent)and)oneHsided)effects.)Biological)Invasions)11:)973–981.)66  Talent)N,)Dickinson)TA)(2005).)Polyploidy)in)Crataegus)and)Mespilus)(Rosaceae,)Maloideae):)evolutionary)inferences)from)flow)cytometry)of)nuclear)DNA)amounts.)Canadian)Journal)of)Botany)83:)1268–1304.)Tas)ICQ,)Van)Dijk)PJ)(1999).)Crosses)between)sexual)and)apomictic)dandelions)(Taraxacum).)I.)The)inheritance)of)apomixis.)Heredity)83:)707–714.)Tepedino)VJ,)Sipes)SD,)Griswold)TL)(2004).)Reproduction)and)demography)of)Townsendia)aprica)(Asteraceae),)a)rare)endemic)of)the)southern)Utah)plateau.)Western)North)American)Naturalist)64:)465–470.)Thompson)SL,)Choe)G,)Ritland)K,)Whitton)J)(2008).)Cryptic)sex)within)maleHsterile)polyploid)populations)of)the)Easter)Daisy,)Townsendia)hookeri.)International)Journal)of)Plant)Sciences)169:)183–193.)Thompson)SL,)Whitton)J)(2006).)Patterns)of)recurrent)evolution)and)geographic)parthenogenesis)within)apomictic)polyploid)Easter)Daises)(Townsendia)hookeri).)Molecular)Ecology)15:)3389–3400.)Van)Dijk)PJ)(2003).)Ecological)and)evolutionary)opportunities)of)apomixis:)insights)from)Taraxacum)and)Chondrilla.)Philosophical)Transactions)of)the)Royal)Society)B:)Biological)Sciences)358:)1113–1121.)Van)Dijk)PJ)(2007).)Potential)and)realized)costs)of)sex)in)dandelions,)Taraxacum)officinale)s.l.)In:)Hörandl)E,)Grossniklaus)U,)Van)Dijk)PJ,)Sharbel)T.)(eds).)Apomixis:)evolution,)mechanisms)and)perspectives,)ARGHGantner:)Ruggell,)Liechtenstein.)pp)215H233.)Verduijn)MH,)Van)Dijk)PJ,)Van)Damme)JMM)(2004a).)Distribution,)phenology)and)demography)of)sympatric)sexual)and)asexual)dandelions)(Taraxacum)officinale)s.l.):)geographic)parthenogenesis)on)a)small)scale.)Biological)Journal)of)the)Linnean)Society)82:)205–218.)Verduijn)MH,)Van)Dijk)PJ,)Van)Damme)JMM)(2004b).)The)role)of)tetraploids)in)the)sexualHasexual)cycle)in)dandelions)(Taraxacum).)Heredity)93:)390–398.)Vielle)Calzada)JHP,)Crane)CF,)Stelly)DM)(1996).)Apomixis:)the)asexual)revolution.)Science)274:)1322–1323.)Whitton)J,)Sears)CJ,)Baack)EJ,)Otto)SP)(2008).)The)dynamic)nature)of)apomixis)in)the)angiosperms.)International)Journal)of)Plant)Sciences)169:)169–182.)Wickham)H)(2011).)The)splitHapplyHcombine)strategy)for)data)analysis.)Journal)of)Statistical)Software)40:)1H29.)67  Appendices(Appendix(A:((Range(wide(survey(of(T.#hookeri(populations(using(pollen(analysis(A.1 Methods(Pollen)analysis)was)performed)on)both)dried)and)fresh)pollen)grains.)Dried)pollen)samples)where)obtained)from)herbarium)mounts)and)from)specimens)collected)by)Ph.)D.)student)Chris)Lee)between)2008)and)2011,)including)new)sites)as)well)as)sites)visited)based)on)past)collections.)From)each)location,)one)to)four)full)plants)(leaves,)flowers,)roots)and)stem))were)collected)for)herbarium)vouchers)and)the)GPS)coordinates)of)the)site)were)recorded)using)a)GARMIN)GPSmap)62st)(Datum)WGS)84).)Fresh)pollen)was)sampled)on)April)2012)from)six)natural)populations)of)Townsendia)hookeri,)in)which)the)ploidy)level)was)known)based)on)previous)collections.)Each)sample)consisted)of)five)disk)florets)that)were)removed)from)one)flower)head,)placed)into)a)labeled)1.5)ml)microtube)and)stored)at)4˚C)within)less)than)six)hours)from)collection.)Pollen)was)obtained)from)four)individuals)in)each)population,)which)had)fully)open)flower)heads)but)unHopen)central)disk)florets,)in)order)to)avoid)collecting)older)pollen)grains.))Pollen)viability)was)tested)using)cotton)blue)in)lactophenol)(Radford)et)al.,)1974),)a)dye)that)stains)callose)with)high)specificity)(Stanley)and)Linskens,)1974))and)has)been)shown)to)accurately)reflect)pollen)fertility)(Mayer,)1991).)Moreover,)lactophenol)was)preferred)to)other)dyes)also)to)ensure)consistency)with)previous)experiments)conducted)on)T.)hookeri)(Thompson)and)Whitton,)2006).)The)preparation)and)mounting)of)the)pollen)involved)removing)six)to)eight)disk)florets)from)dried)inflorescences,)including)fieldHcollected)plants)and)herbarium)mounts,)and)placing)these)into)1.5ml)centrifuge)tubes.)This)first)step)does)not)apply)to)fresh)pollen)samples,)which)were)already)kept)in)1.5)ml)tubes.)20)µL)of)cotton)blue)and)10)µL)of)distilled)water)were)added)to)each)tube)and)the)68  tubes)were)vortexed)for)one)minute)to)release)pollen)into)suspension.)15)µL)of)the)resultant)suspension)was)placed)on)a)slide.)The)slide)was)covered)immediately)with)a)coverH)slip)and)the)edges)were)sealed)with)nail)polish.)Generally,)the)pollen)was)observed)within)a)few)hours;)however,)we)noticed)that)slides)viewed)immediately,)as)well)as)days)or)weeks)after)preparation,)gave)consistent)results.)At)least)300)grains)were)counted)in)each)slide)and)pollen)grains)with)fully)stained,)regularly)shaped)cytoplasm)were)scored)as)viable,)while)pollen)units)that)were)a)lighter)blue)in)color,)translucent)and)irregularly)shaped)were)considered)inviable.)The)size)of)the)pollen)was)assessed)by)measuring)the)cytoplasm)diameter)of)twenty)viable)pollen)units)per)slide.)Pollen)counts)and)diameter)measurements)were)obtained)using)a)Nikon)Eclipse)80i)light)microscope,)at)10x)and)40x)magnification,)respectively.)The)proportion)of)stainable)pollen)and)the)mean)grain)size)were)recorded)and)the)graphical)analysis)of)the)data)was)conducted)using)R,)version)3.0.3)(R)Core)Development)Team,)2014).)) )69  A.2 Results( Figure(A.2.1(Plot)of)mean)cytoplasm)diameter)of)viable)grains)by)mean)pollen)stainability,)representing)diploid)(dark)grey)circles))and)polyploid)(white)circles))cytotypes.)For)each)ploidy)category,)mean)stainability)(±standard)error))and)mean)viable)pollen)size)(±standard)error))were)found)to)be)equal)to:)82.81±2.63)%)and)22.31±0.25)µm)for)diploids)(n=24);)25.73±2.3)%)and)34.75±0.86)µm)for)polyploids)(n=34).) ! !Mean pollen diameter (µm)Mean pollen stainability (%)2040608010020 25 30 35 40 45●●●●●●●●●●●●●●●●●● ●●●●●●● ●●●●●●●●●●●●●●●●●● ●●●● ●●●●●●●●●● Ploidydiploidpolyploid●●70  Appendix(B:((Range(wide(survey(of(T.#hookeri(populations(using(flow(cytometry(B.1 Methods(TwentyHfive)populations)of)T.)hookeri)were)located)based)on)past)collections,)namely)herbarium)specimens)and)sites)described)by)Chris)Lee)between)2008)and)2011.)The)survey)was)limited)the)portion)of)the)range)outside)Yukon)(from)southern)British)Columbia)to)Colorado).)All)the)localities)were)visited)in)June)2012)and)leaf)samples)from)at)least)three)different)individuals)were)obtained)in)each)population,)with)the)exception)of)two)sites)in)Wyoming,)where)only)one))and)two)plants)were)located)and)sampled.)The)leaf)material)was)stored)at)ambient)temperature)in)paper)envelopes)containing)silica)gel.)Within)four)to)five)weeks)of)collection,)the)dehydrated)tissue)was)removed)from)silica,)transferred)to)2)ml)screw)cap)microtubes)and)stored)in)a)H80˚C)freezer.)The)samples)were)analyzed)via)flow)cytometry)between)November)2012)and)April)2013)following)the)protocol)describe)in)section)2.2.4.)) )71  B.2 Results(!Figure(B.2.1(Genome)size)(2CHvalue))of)68)samples)representing)25)populations)of)the)Easter)Daisy)(samples)from)the)same)population)always)cluster)together).)The)values)were)estimated)performing)flow)cytometry)on)silicaHdried)leaf)tissue.)Samples)were)assigned)to)three)different)ploidy)levels:)diploid)(2Cx;)red)circles),)triploid)(3Cx;)blue)circles))and)tetraploid)(4Cx;)light)blue)circles).)For)each)ploidy)category,)mean)2CHvalue)(±standard)deviation))was)found)to)be)equal)to:)13.02±0.5)pg)for)diploids)(n=23),)18.34±0.89)pg)for)triploids)(n=33))and)22.58±1.28)pg)for)tetraploids)(n=12).) 2C−value (pg)11 13 15 17 19 21 23 25●●●●● ●●●●●●●●●●●●●●●● ●●●● ●●●●●●●●●●● ●●●● ●●●●●● ●●● ●●● ●●● ● ●●●●●●72  Appendix(C:((Results(from(the(coJground(test((FCM)(Results)from)the)coHground)trial)(section)2.3.2))including:)identification)numbers)of)the)populations)of)origin)(ID)p.),)ploidy)of)the)populations)of)origin)based)on)the)results)of)pollen)analysis)(Ploidy)p.),)ID)of)the)samples)tested)via)flow)cytometry)(ID)s.),)average)2CHvalue,)standard)deviation)(std.)dev.))and)ploidy)(DNA)ploidy))obtained)via)flow)cytometry.)DNA)ploidy)categories)are)defined)based)on)the)work)presented)in)Appendix)B.)ID(p.( Ploidy(p.( ID(s.( Mean(2CJvalue((pg)( std.(dev.( DNA(ploidy(2019) 3x) 19C4) 18.847) 0.015) 3Cx)2019) 3x) 19C6) 19.271) 0.018) 3Cx)2019) 3x) 19C8) 19.052) 0.067) 3Cx)2031) 3x) 31C1) 17.134) 0.048) 3Cx)2031) 3x) 31C2) 19.045) 0.010) 3Cx)2033) 2x) 33C1) 12.555) 0.038) 2Cx)2033) 2x) 33C3) 13.328) 0.099) 2Cx)2030) 2x) 30C3) 13.412) 0.018) 2Cx)2030) 2x) 30C5) 13.176) 0.258) 2Cx)2030) 2x) 30C7) 12.810) 0.069) 2Cx) 

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