Pulsed evolution shaped extant angiosperm pollen disparity

Domain: earth_science
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Pollengrains,themalegametophyteofseedplants,areenclosedwithin
sporopolleninwallsthatyieldremarkablestructuraldiversityandexceptional
preservation,makingtheminvaluableproxiesforpastplantdiversity.Despite
theirimportance,thediversificationpatternsofangiospermpollenarenot
wellquantifiedorunderstood.Byintegratingagenus-levelpollentraitdataset
withatime-calibratedphylogeny,wequantifiedtheevolutionarydynamicsof
angiospermpollen,includingitsmorphospaceoccupation,disparity,and
ratesofmorphologicalevolution.Ourresultsrevealthatangiospermpollen
disparityexpandedthroughtwomajorpulsedincreasesduringtheMidCre-
taceousandPaleogeneperiodsmarkedbysignificantenvironmentalshiftsand
correlatedwithkeypolleninnovations.Thesefindingsarerobustunder
varyinghypothesesofcrownangiospermageandhighlightasynergistic
interplaybetweenevolutionaryinnovationandkeyecologicalopportunities,
ratherthanasingularearlyburstofdiversification.Basedoninferencesfrom
extantmorphologies,ourresultsshowthattheinitiallylowdisparityinearly
angiospermevolutionwasfollowedbyarapidMidCretaceoussurge,pro-
vidingcriticalcontextforinterpretingpatternsobservedintheangiosperm
fossilrecord.Theseresultsunderscorethepowerofpollendisparityintracing
angiospermradiationandunderstandingthediversificationofEarth’smulti-
cellularlife.

Introduction
Pollengrains—themalegametophytesofseedplants—exhibitaston-
ishinglyintricatestructureswithinmicroscopicdimensions,captivat-
ing scientists and artists for centuries1–5. The outer wall (exine) of
pollengrainsprovidesabarrieragainstchemicaldamageandencases
themalegeneticmaterial,withareasofreducedexineformingaper-
turesthatarethestartingpointsforpollentubegrowthandthereby
pollination6.Whilepollengrainsaredesignedtoprotectandtransfer
male genetic material, the morphological variation in both the
apertures and the wall makes them among the most diversified
microstructuresinnature(Fig.1)3,4,7.Thisdiversityisthoughttopro-
vide varying harmomegathic stress systems – the folding of pollen
grains to accommodate cellular volume changes due to hydration
variations(Wodehouseeffect)2–andisbelievedtoplayaroleinthe
efficiency of interactions with pollinators and in pollen-stigma
interactions6,8,9. The developmental origins of pollen morphological
diversity (disparity) have been explored in numerous taxa with key
findingsbeingtheinfluenceofminorvariationsinmicrosporemeiosis
on pollen symmetry and aperture position, and the role of self-
assemblyandphaseseparationintheultrastructureoftheexine10–13.
Understandinghowthediversityoforganismalformsarisesisa
crucialtopicinevolutionarybiology14,15,andthestudyofangiosperm
pollentraitevolutionwithinabroadevolutionarycontexthasapar-
ticularly rich history1,3,16,17. Pollen traits played a pivotal role in the
morphological diagnosis, through cladistic analysis, of the eudicots
(i.e., ‘the tricolpate clade’)—a monophyletic group comprising
approximately75%ofextantangiospermspecies—distinguishedbythe
synapomorphy of equatorially tricolpate pollen18,19. This was later
regardedasakeyinnovationinearlyangiospermevolution11andthe
monophylyoftheeudicotswassoonconfirmedusingmolecularsys-
tematics.Meanwhile,owingtoitswidespreaddistribution,taxonomic
distinctiveness,abundance,andexceptionalpreservationpotentialin
sedimentaryrocks,pollenhasbeenextensivelyemployedintracing
pastvegetationandclimatechangesonEarth7,20–22.However,despite
thisimportance,thelarge-scalepatternsofangiospermpollendiver-
sification remain poorly quantified and understood. This lack of a
quantitative framework limits—and may even distort—our under-
standingofangiospermradiation,particularlyregardingdeepevolu-
tionaryprocesses.

Morphological diversity is one of the most intuitive ways to
quantifymorphologicalvariationswithincladesandhascontributed
substantiallytodocumentingthedynamicsofevolutionaryhistory—
sometimesreferredtoasthe“kinetics”ofbiologicaldiversity23.Ina
paradigm rooted in zoology24–27, early stages in the evolution of a
groupareexpectedtofeaturelessconstrainedgeneticanddevelop-
mental systems coupled with abundant ecological opportunities,
leadingtoaswiftpeakindisparity.Lupia’s28quantitativeanalysisof
fossilpollenmorphologiesduringNorthAmerica’sCretaceousPeriod
andPalaeoceneEpochdemonstratedarapidriseinEarlyCretaceous
pollen disparity, often cited as a typical example supporting this
model.PollendisparityintheAsterales—thelargestorderofangios-
perms—further corroborates this pattern29. A plausible evolutionary
explanationforthismodelisthattheearlysuccessofangiospermswas
intricately linked to their capacity for rapid reproduction30–32, with
selection favouring short-lived pollen grains capable of quick
germination32. This process likely drove a series of morphological
changes,particularlyintheaperturestructure.Interestingly,palyno-
logical observations of extant early-divergent angiosperms confirm
thepresenceofrelativelydiverseaperturedesigns(Fig.1)16,33.

Theearlymaximumdisparitymodelfacesscrutinyfromseveral
disparity studies, which suggest that morphological radiations may
occur at any point in a clade’s history, with their appearance con-
tingentonthetemporalandtaxonomicscalesoftheanalysis34,35Phe-
notypicdisparityresearchacrossanimals,plantsandfungiindicates
sporadic growth patterns, portraying early pulse events like the
Cambrian explosion as mere snapshots of fossilizable traits, inade-
quateforexplainingoverallphenotypicdisparity36–38.Sincedisparity
studieshavepredominantlyfocusedonanimals,andconsideringthe
distinctiveevolutionarytrajectoriesofanimalsandplants—sometimes
likenedto“dancingtoadifferentbeat”39—itremainsunclearwhether
similar disparity patterns exist in plants. Beyond pollen, studies by
Oystonetal.40haveshownthatmostplantcladesreachedtheirmor-
phologicaldiversityzenithsoonafterdiverging;angiospermflowers
achievedtheirdisparitypeakintheEarlyCretaceous41,whereaswood
functional disparity only began its rapid ascent in the Mid
Cretaceous42.

Extensiveandreliablefossilpollenrecordswouldundoubtedlybe
the best data sources for quantifying angiosperm pollen disparity.
However,findingreliableangiospermfossilsfromtheEarlyCretaceous
andearlierishighlychallenging,andtaxonomicuncertaintyinmany
microfossils limits their integration into phylogenetic frameworks,
therebyconstrainingtheiruseinmacroevolutionaryanalyses21,22,43–47.
Forinstance,themostreliablecrownageforangiospermsiscurrently
establishedusingBarremian–Aptiantricolpatepollen,butthistypeof
pollenshoulddefinethecrownageofeudicots,thusunderestimating
thetrueageofcrownangiosperms48,49.Similarly,theprevailingten-
tative candidates for pre-Cretaceous angiosperm fossils also derive
from pollen, dated to the Middle Triassic, though the reproductive
structureproducingthispollenremainsunknown50.Moreover,earlier
fossil pollenworks,suchasthosebyDoyle51andLupia28, were con-
strained by a limited understanding of evolutionary relationships
among groups and were restricted in geographical and temporal
extent. Given these challenges, there is a clear need to reevaluate
angiosperm pollen disparity in a modern phylogenetic context. By
integratingtime-calibratedphylogenetictrees,wesituatethepollen
morphospace within a broader evolutionary context, providing
insights into morphological evolution and enhancing our under-
standingofangiospermradiation.

Here, we compile a genus-level dataset of extant pollen
morphologiesandatime-calibratedphylogenytoexplorethelarge-
scale evolutionary dynamics of angiosperm pollen, including mor-
phospaceoccupation,disparity,andratesofmorphologicalevolution.
To examine how different structural domains contribute to overall
pollen disparity, we defined three analytical subsets: pollen grain
shape (shape), aperturepattern (aperture), and exine structure and
ornamentation (exine). These subsets were each analysed indepen-
dently,alongsidethefullmatrix,toprovideamodularframeworkfor
interpretingpollendisparity.Inaddition,weexaminehowpollendis-
paritycorrelateswithpollinationtype,genomesize,andtaxonomic
diversitytoidentifypotentialrelationshipsbetweentheseparameters.
This study aims to elucidate the evolutionary mode and tempo of
pollen disparity, assessing its compatibility with the early maximal
disparity model and offering palynological insights into the evolu-
tionarydynamicsofangiospermdiversification.

Results
Wedevelopedadiscrete-codedmatrixof20pollencharactersspeci-
ficallydesignedtocapturethefundamentalvariationsinpollenmor-
phology,withgeneraastheterminalunits(SupplementaryData1and
Supplementary Table 2). These characters are grouped into three
major morphological categories: shape (characters 1–6), aperture
(characters7–13),andexine(characters14–20;referringexclusivelyto
the stratified layers of the pollen wall). These groupings are con-
sistentlyusedthroughouttheresultsandaredefinedinSupplemen-
taryTable2,whichalsoprovidesdetaileddescriptionsofall20pollen
charactersandtheirdiscretestatesusedinthisstudy.Weensuredthe
proportionofmissingdatawaskeptbelow5%(includinginapplicable
codes) to mitigate the adverse effects of missing data, and poly-
morphicstatesaccountedforapproximately6.2%ofthegenus-level
codings.Fortheselected1,880genera,wereconstructedamaximum
cladecredibility(MCC)treethatencompassesall64ordersand434
recognisedfamiliesofangiosperms(Fig.1).Thetopologyisbroadly
congruentwithrecentplastome-basedphylogeneticframeworks52,53,
althougharecentnuclearphylogenomicstudy50suggestssomealter-
nativerelationships,particularlywithintherosidclade.Ourtopology
supports relationships [Chloranthales [magnoliids [monocots [Cer-
atophyllales+eudicots]]]] within the mesangiosperms (Fig. 1). All
ordersandfamilieswererecoveredasmonophyleticinourphylogeny,
followingtheexclusionofroguetaxatostabilisetreetopology.

Current consensus maintains that fossil-calibrated molecular
datinganalysescannotreliablyresolvethecrownageofangiosperms,
thoughtheytypicallyemploymaximumageconstraintsonthecrown
nodeofangiospermsbasedonpriortemporalboundaries,ultimately
producing alternative chronological frameworks (young vs. old
angiosperm time trees)54–56. Our date trees show closer congruence
withtheplastidphylogenomicangiosperm(PPA)treeofLietal.52than
with other recent dating studies, likely reflecting shared time-
calibration strategies despite distinct molecular datasets (Fig. 1 and
SupplementaryFig.1).Temporaldiscrepanciesamongourthreedated
treesprimarilystemfromdifferentialmaximumageconstraintsonthe
crown node of angiosperms, with significant stem age variations
observedinthethreeordersoftheANAgradeandfivemajormesan-
giosperm clades (spanning Early Cretaceous to Early Triassic) (Sup-
plementaryFig.1).Notably,ourdatedtreesalignmorecloselywiththe
‘youngtree’scenarioatordinalandfamiliallevels,incontrastto‘old
tree’scenariosthatplacemostordinaldiversificationsintheTriassic—a
timeframethatconflictswithcurrentpaleobotanicalevidence22,44.

We carried out both Non-metric Multidimensional Scaling
(NMDS)andPrincipalCoordinatesAnalysis(PCoA),withNMDSprov-
ingmoreeffectiveforvisualisingandinterpretingmorphospaceinthis
context(Fig.2).ThetwoNMDSaxesshowedstrongcorrelationwith
the first two axes of the PCoA (Spearman’s r>0.84, P<0.001),
accountingfor21%and14%ofthevariance,respectively.Moreover,in
ourassessmentofthereliabilityofordination-basedmetrics,NMDS-
basedMeanPairwiseDistance(MPD)showedstrongconcordancewith
values derived from the raw distance matrix (Pearson’s r=0.957,
P<0.001),indicatingminimaldistortion.Bycontrast,MPDbasedon
PCoAcoordinateswaslessstronglycorrelated(r=0.737,P=0.0063)
(SupplementaryFig.2).Correlationtestsamongthedistancematrices
ofthesubsetsrevealedthatshapeandaperturewereweaklycorrelated
(Mantel’s r=0.285, P=0.0099), whereas the correlations between
exineandtheothertwocomponentswereveryweak(r=0.104-0.136,
bothP=0.0099).

Pollenmorphospaceoccupation
TheNMDSandPCoAordinationsrevealedthattheangiospermpollen
morphospaceformsacontinuous,cloud-likedistribution(Fig.2a).In
these ordinations, Axis 1 showed the strongest association with
polarity(shape)andthepresence/absenceofendoaperture(aperture),
while Axis 2 was associated most strongly with tectum sculpture
(exine) and the presence/absence of supratectal elements (exine),
highlightingtheimportanceofthesefourtraitsincharacterisingpol-
len disparity (Supplementary Fig. 3 and Supplementary Table 2).
Althoughtheseaxesarebasedondistanceratherthanrealbiological
variables,correlationswithoriginalpollentraitsallowingustoinferthe
gradientsthateachaxisrepresents(SupplementaryFig.3).Basedon
the sum of variances (SOV) across NMDS axes, the empirical mor-
phospaceofallextantgeneraencompassesapproximately49.0%of
thetheoreticalmorphospace’stotalvariance(Fig.2c).Whenmeasured
usingthesumofranges(SOR)acrossaxes,theoccupiedproportionis
substantiallylarger,reaching~89.6%(Fig.2c).Thisoccupiedregionis
concentratedtowardthenegativeendofAxis1,whichcovariesmost
strongly with traits such as pollen size (shape) and the presence/
absence of tectum (exine). Notably, when restricting the empirical
morphospace to the central 95% of genera (excluding extreme
outliers),theoccupiedspacecontractstoonly~32%ofthetheoretical
varianceand~51%oftherange(Fig.2c).

Under a within-clades analysis,eudicots asa wholeoccupy the
largest morphospace area (SOR) among angiosperms, while within
eudicots,fabids,representoneofthelargestsubcladesintermsofSOR
across all four dimensions—overall disparity, shape, aperture, and
exine(SupplementaryFig.2).Thesoleexceptionistheexinesubset,
whereeudicotshaveasimilarrangevaluetomonocotsintheNMDS
results,andasmallerrangeinthePCoAresults.Withintheeudicots,
fabids,theothersuperasterids,andlamiidsoccupythelargestareas
(SupplementaryFig.2).Moreover,thesethreesubgroupsachievethe
highestdisparitylevelsintheexine,aperture,andshapesubsets.Sum
ofVariances(SOV),MeanPairwiseDistance(MPD),andMeanDistance
toCentroid(MDC)displayed similarsizetrends.Intermsofoverall
pollen disparity, monocots and the other superasterids exhibit the
highest disparity levels among angiosperms and eudicots, respec-
tively. In shape, magnoliids and the other superasterids stand out,
whileforapertures,itismonocotsandtheothersuperasterids.Inthe
exinesubset,monocotsandfabidsshowthehighestdisparity.Details
ofthecomparativeresultsoftheseindicesareshowninFig.2band
SupplementaryFigs.2,4–7.

A k-means clustering analysis divided the pollen morphospace
intofourclusters,witheudicotsformingexclusiveclustersforshape
and aperture subsets, while the distribution was even in the exine
patternamongmajorgroups(SupplementaryFig.8).Disparitydiffers
significantly among angiosperm groups (Kruskal-Wallis test:
χ2=4669.6,df=4,P<0.001)andeudicotgroups(χ2=6149.2,df=6,
P<0.001) (Fig. 2). Using the subgroup omission method, we found
thatcommelinids,theothermonocots,andfabidsmadethehighest
contributionstooverallangiospermpollendisparity,whiletheANA
grade, Chloranthales, and the other superasterid made the lowest
contributions (Supplementary Fig. 5). The PERMANOVA analyses
revealed a statisticallysignificantseparationin pollen morphospace
occupation among the major angiosperm groups and among the
majoreudicotgroups(seeSupplementaryData2fordetail),indicating
that most subgroups possess distinct trait combinations. Notably,
morphospaceseparationwasmorepronouncedbetweeneudicotsand
other groups, whereas the ANA grade and Chloranthales were not
clearly distinguished from the other non-eudicot groups (Supple-
mentary Data 2). Compared with the NMDS results, PCoA showed
greater discriminating power: all eudicot groups exhibited a clear
separationintheirmorphospaceoccupation(SupplementaryFigs.6,7
andSupplementaryData2).

Theobservedpatternsofmorphospaceoccupationareshapedby
adetectablephylogeneticstructure.AManteltestrevealedamoderate
butsignificantcorrelationbetweenthepollenmorphologicaldistance
matrixandthephylogeneticdistancematrixderivedfromthetime-
calibratedtree(Mantel’sr=0.397,P=0.0099),indicatingthatevolu-
tionarytimecontributestooverallpollendisparity.Phylogeneticsig-
naltestsusingBlomberg’sKfurthersupportedthisview:Axis1ofboth
NMDSandPCoAordinationsexhibitedstatisticallysignificantsignals
(K=0.10and1.03,respectively;P<0.001)(SupplementaryFig.9).
TheC1 convergenceanalysisforallgenuspairsrevealedamedian
C1valueof0.231,indicatingamoderatereductioninancestralmor-
phological divergence. The C1 distribution (Supplementary Fig. 10)
exhibitedabimodalpatternwithaprimarypeakatlowC1values(near
zero),reflectingwidespreadmorphologicaldivergence,andadistinct
secondarypeakatC1=1.0,representingextrememorphologicalcon-
vergence. In total, 203,760 genus pairs (5.77% of all pairs) demon-
stratedhighconvergence(C1≥0.90).Thetaxonomicrankanalysisof
these highly convergent pairs (Supplementary Fig. 10) showed the
majorityoccurredatG1(70.1%)andG2(17.6%)ranks,whereasonlya
smallfractionwasobservedatotherranks(Family:6.1%;Order:5.1%;
Subclass:1.1%),indicatingthathighmorphologicalconvergencearises
amongtaxonomicallydistantlineages.Here,G1andG2ranksrepresent
twoinformaltaxonomicranksbetweenorderandsubclass,withspe-
cificdivisionsdetailedintheMethodssection.

Ratesofmorphologicalevolution
Ratesofmorphologicalcharacterchangewerequitelow(<;0.1char-
acter state changes/Ma) until the Neogene, but increased markedly
thereafter,reaching0.21characterstatechanges/MaintheNeogene
and 0.58 character state changes/Ma in the Quaternary (Fig. 3 and
SupplementaryFig.11).Intestsofbranchrates,significantcharacter
statechangesweredetectedin599branches,andamongthem,the150
internalbranchesaremarkedinFig.3.Intestsofmorphologicalevo-
lutionratesbasedonoverallpollentraitsandtheaperturesubset,the
topfifteenfastest-evolvingbranchesforeachtraitsetaremarkedin
Fig. 3. Three notable branches—the ones leading to the eudicots,
superrosids, and asterids—appear in both rankings (see Fig. 3 and
SupplementaryTable3).Amongthese,thebranchleadingtoeudicots
showed unambiguous state changes from heteropolar and mono-
aperturategrainstoisopolarandtricolpategrains,whilesuperrosids
and asterids displayed the emergence of endoapertures (Fig. 3 and
Supplementary Table 3). Meanwhile, the branch leading to the
Nitrogen-fixingCladeexhibitedthehighestrateofpollenmorpholo-
gical change (1.66 character state changes/Ma)—a particularly note-
worthyfinding(Fig.3andSupplementaryTable3).

Inratetestsforindividualtraits,tectumsculpture,size,outlinein
polar view (shape), supratectal element presence/absence, and
endoaperture presence/absence (aperture) were the five fastest-
evolving traits, with tectum sculpture evolving at a rate approxi-
mately four times that of the second fastest, size (1.74 versus 0.45
characterstatechanges/Ma)(SupplementaryFig.11).Meanwhile,the
fiveslowest-evolvingtraitswerebasicshape(shape),exinepresence/
absence(exine),macro-ornamentationtype(shape),tectumpresence/
absence,andinfratectumtype(exine)(SupplementaryFig.11).Apart
fromthepresence/absenceofendoaperture,aperturetraitsgenerally
exhibited moderate evolutionary rates. Further analysis confirmed
that rates of morphological evolution across the three blocks were
fastest for the exine, followed by aperture and shape (Supplemen-
taryFig.11).

Pollendisparitythroughtime
Weanalysedtimeslicesfromourdatedtreecoveringthreepossible
crownagescenariosforangiosperms:LateTriassic,EarlyJurassic,and
EarlyCretaceoustothepresent.Theresultsindicatethat,underthese
different crown age hypotheses, variations in pollen disparity over
timeprimarilypertaintotheinitialexpansionofpollenmorphospace
during the establishment of the clade (Fig. 4 and Supplementary
Fig.12).However,aconsistentpatternemerges:underallscenarioswe
seearapidincreaseindisparityduringtheAlbian–Cenomanianstages
(~20Ma)oftheCretaceous,dominatedbychangesinaperturepat-
terns.Thissurgeisfollowedbyaprolongedperiodofstability—orvery
slow growth—during the Late Cretaceous, then a steady increase
beginningattheCretaceous–Paleogenetransition,contributedbyall
three subsets of characters (aperture, exine and shape), reaching a
peak by the Oligocene (Fig. 4). This evolutionary pattern remains
consistentacross100randomsubsamples,eachwithasamplesizeof
1000,andprovesstableundervaryingrarefactionlevelsanddifferent
size-dependent indices, underscoring the robustness of the dataset
(SupplementaryFig.12).

Byplottingthedisparitythroughtime(DTT)foreachofthethree
subsets of characters, we can observe the varying contributions of
eachsubsettotheoveralldisparityatdifferentstagesofangiosperm
evolution(Fig.4).Thethreesubsetsexhibitdistinctpatternsofdis-
parityaccumulation,withthedisparityinaperturepatterns,particu-
larly, experiencing an approximately fivefold increase across the
Albian–Cenomanianboundary(Fig.4).

Correlations
Ourfindingsrevealthatpollendisparityisnotcorrelatedwithtaxo-
nomic diversity in angiosperms (Mantel’s r=-0.01, P>0.5; Supple-
mentaryFig.13).However,weobserved weak but significant
correlationswithgenomesize(Mantel’sr=0.104,P<0.001;Supple-
mentaryFig.13).Althoughthecorrelationbetweenpollinationmode
andtheprimarytwoaxesoftheNMDSandPCoAordinationwasweak
(Pearson’sr=0.1–0.24,P<0.001),statisticallysignificantseparations
wereobservedamongmostpairsofpollinationmodes(e.g.,abiotic
versusbiotic,windversusinsect)(SupplementaryData3).Notably,the
separationbetweeninsectandvertebratepollinationwasnotstatisti-
callysignificant,asindicatedinSupplementaryFigs.14,15andSup-
plementaryData3.

Methods
Phylogeneticreconstruction
Giventhesignificanceofpollentraitsinangiospermsystematics,our
goalwastobalancetherepresentationofdiversepollenmorphologies
with comprehensive phylogenetic coverage. Consequently, we sam-
pled 1926 genera, including 1880 angiosperm genera across 434
familiesand64orders,and46gymnospermgeneraasoutgrouptaxa.
Wechosetousegeneraasterminalunits—achoiceconsistentwith
standard practice in both palynology and macroevolutionary
studies43,100.Generaarewidelyregardedasevolutionarily‘real’units,
often reflecting shared evolutionary trajectories and coherent mor-
phologicalpatternsamongtheirconstituentspecies100,101.Genus-level
coding reduces species-level sampling bias while still capturing
meaningfulproxiesforecologicalandmorphologicaldiversity100.This
levelofsamplingisalsowell-suitedtopalynologicaldatasets,owingto
the relatively conservative nature of pollen variation within genera
(andsometimeswithinfamilies).

Recent molecular supermatrix studies have shown that most
currently accepted angiosperm genera are now well circumscribed
and generally supported as monophyletic102,103. Given the macro-
evolutionary scale of our study, potential non-monophyly of some
genera is unlikely to affect our main conclusions. Nevertheless, to
ensure taxonomic consistency, we cross-validated the status of all
sampledgeneraagainstglobaldatabases,includingPlantsoftheWorld
Online(POWO;accessedMay2021)andtheCatalogueofLife(COL;
accessedMay2021).Generawithambiguousorunresolvedtaxonomic
placements were excluded from subsequent analyses. Although
genomicdatacouldprovideamorerobustangiospermbackbone,we
optedforagene-samplingandcompositetaxonapproachtoensure
thoroughcoverageofallrecognisedfamiliesandtheirrepresentative
infra-familial branches (predominantly subfamilies). While recent
phylogenomic studies, such as Zuntini et al.56 have proposed alter-
native ordinal relationships based on nuclear dataset, particularly
withinrosids,wefollowedtheordinalandmajorcladedefinitionsof
APG IV104, which remain widely accepted and facilitate consistency
across comparative analyses (Supplementary Data 1). Infra-familial
subdivision is mainly based on the Angiosperm Phylogeny website
(http://www.mobot.org/MOBOT/research/APweb),covering111famil-
ies.Thecompositeterminalstrategyallowsforthesupplementationof
terminal unit sequences with sequences from the same genus, sig-
nificantlyincreasingthegeneticmarkercoverageforeachgenus.Our
samplingapproachoffersseveraladvantages:(i)extendedsamplingat
thesubfamiliallevelusinggeneraasterminalsenhancesourcaptureof
morphologicalvariations;(ii)thecompositetaxonapproachgreatly
improvesphylogeneticanalysisaccuracybyreducingmissingdata105;
(iii)thismethoddoesnotcompromisehigher-levelrelationships,asit
doesnotfocusontheevolutionaryhistorywithingenera.

For the sampled genera, we extensively searched GenBank for
commonlyanalysedgeneticregionsacrossallthreeplantgenomes,
including plastid genes (atpB, matK, ndhF, rbcL, rpoC2, and rps4),
mitochondrialgenes(atp1,matR,nad5,andrps3),and nuclearribo-
somalgenes(18Sand26S).Accessionnumbersandassociatedtaxo-
nomic information for all genera included in the final dataset are
providedinSupplementaryData4.The12-locusdatasetwasmeticu-
louslycuratedthroughiterativescreeningofindividuallociandcon-
catenatedmatricestoidentifyandaddressanyroguetaxa,ensuring
thatthesequencesofthecompositeterminalsoriginatedonlyfrom
monophyleticgenera.Aftermanuallyinspectinginitialtrees,wecon-
catenatedthelociandusedRAxMLtoinferamaximumlikelihood(ML)
tree. We then applied penalised likelihood, as implemented in the
programme treePL106, to the topology from the RAxML analysis for
moleculardating,utilising56fossilcalibrationpointsdescribedinLi
etal.52andsettingasmoothingvalueof0.01.Wefurtherconstrained
ourestimatesbyspecifyingdates(139.4Maand209Ma)forthecrown
ageofangiospermstoexplorepotentialeffects52,107.

Morphologicaldataset
Pollen character scoring was based on based on multiple sources,
including experimental observations, PalDat database (https://www.
paldat.org),andthepalynologicalliterature(SupplementaryData5).
All records were reassessed and recoded according to the coding
strategies outlined here (Supplementary Table 2 and Supplemen-
taryData1).

To effectively characterise the myriad features of pollen, paly-
nologistshavedevelopedaricharrayofcomplexterminology.Punt
etal.108documented588pollenterms,recommending349foruse—a
striking figure reflecting the minute structural details of pollen. In
paleopalynology, “pollen type” is commonly employed to describe
pollengrains,provingvaluableinidentifyingisolated,dispersedfossil
pollen.However,thisapproachislesssuitedtoourstudy.Phylogenetic
comparativemethodsprioritisesharedtraits,whereasincorporating
excessivefeaturesintroducesexcessivemissingdataandcomplicates
the weighting of numerous morphological variables. In line with
recommendationsofWortleyetal.98andPuntetal.108,westandardised
terminology based on widely accepted glossaries (Supplementary
Table2).Whenoriginaldefinitionswereinconsistentoroutdated(e.g.,
duetoLM-onlyhistoricalusage),werecodedcharacterstatesbasedon
contemporaryinterpretationssupported bySEMimagesand recent
palynologicalreviews.Descriptionsofcharacterstateswereextracted
directlyfrompublishedpollenmicrographs,supplementedbytaxo-
nomiccommentswhennecessary.Thisdirectvisualscoringapproach
avoidsrelianceonover-summariseddatabaserecordsorambiguous
traitlabels.

FollowingWortleyetal.98,weprioritisedpollencharactersthatare
(i) taxonomically widespread, (ii) variable at the ordinal or intra-
familiallevel,and(iii)observableunderLMand/orSEMwithminimal
ambiguity.Ultimately,weselected20discretepollentraitsthatreflect
essential aspects of pollen variations. Complex characters were
decomposedintomorebasicandconsistentlyscorablecomponentsto
minimise subjectivity. We acknowledge that categorisation of char-
acters is inherently constrained by both technical and conceptual
factors109.Traitobservabilitydependsnotonlyonthematurityofthe
pollengrainbutalsoonthemodeofobservation(e.g.,LMvsSEMvs
TEM),samplepreparation(e.g.,acetolysis,hydrationstate),andsub-
jectiveinterpretation98,109.

Weuseddiscretecharacters,ratherthancontinuouscharactersor
geometricmorphometricapproaches,tocapturepollenmorphology,
becausethisallowedustoincorporatethefullrangeofmorphological
features,includingaperturetypeandexinestructure,inonecoherent
framework40. Classical and geometric morphometrics (e.g., length/
width ratios, elliptic Fourier) are powerful within relatively homo-
geneousmorphospacesbutlosetractionasdisparitybroadensacross
higher taxa40. Discrete characters, by contrast, allow comparison
acrossthefullbreadthofpollenmorphologiesandcapturebiologi-
callyandphylogeneticallyinformativefeatures(aperturearchitecture,
exinestratification,polarity)thataredifficulttoreducetocontinuous
metrics.Theflexibilityofdiscretecoding—despiteitscoarsergranu-
larity—remainsadvantageousforcapturingthefullextentofdisparity
inlarge-scaleevolutionaryanalyses15,40.

Torepresentwithin-genusvariation,weusedpolymorphiccoding
andretainedallobservedstateswithoutmodification(e.g.,0&1&2).As
standard in discrete-character matrices, our use of polymorphism
excludesintraspecificvariationattributabletoontogeny,geography,
orplasticity110,111.Rareorambiguousstateswereconservativelycoded
as missing. This approach avoids imposing unverifiable frequency
assumptions (as in “democratic coding”) while preserving observed
variation. Finally, the matrix explicitly distinguishes ‘not applicable’
from missing entries, providing a consistent basis for downstream
morphospaceanddisparityanalyses.Wefurthersegmentedthedata
intothreesubsets:(1)shape(characters1–6);(2)aperture(characters
7–13);and(3)exine(characters14–20)(SupplementaryTable2).

Distancematrixandordination
Wefollowedthe“preordinationancestralstateestimation”(pre-OASE)
approachproposedbyLloyd112.Thismethodfirstreconstructsances-
tralcharacterstatesforthenodesofthephylogenyusingmaximum
likelihood estimation, then calculates morphological distances to
generatepairwisedistancematrices,andfinallyordainsthedistances
toproduceamorphospace.Pairwisedistanceswerecomputedwitha
metric that accommodates polymorphisms (maximum observable
rescaleddistance,MORD),treating differences conservatively(mini-
mumdifference)sothatapolymorphicstateiscomparedtotheclo-
sestobservedcounterpart.Toassesstherobustnessofthematrices
underdifferentdistancemetricsandcharacterdependencyvalues112,113,
weevaluatedninedistancematricesofpollendisparityusingvarious
metrics: raw Euclidean distance (RED), Wills’ generalised Euclidean
distance (GED), Gower’s coefficient (GC), maximum observable
rescaleddistance(MORD),andalternativealphavalues(0,0.5,and1),
which represent the impact level of secondary characters on the
distance114.ThestabilityofthesematriceswastestedusingaMantel
testwith99permutations.AfterexcludingtheleastcorrelatedGED
metrics,thepairwisecorrelationcoefficient(r)rangedfrom0.828to
0.998.WeultimatelychosetheMORDdistancemetricwithanalpha
valueof0.5asourmaindistancematrix,whichisanextensionofthe
GCandiseffectiveinhandlingpolymorphictraitstates113,115.Giventhe
highcorrelationsamongalternativemetrics,ourdownstreamresults
areexpectedtoberobusttothechoiceofdistancemeasure.Allthese
functionswereimplementedinCladdisver.0.63113inR116.

Wethenappliedtwoordinationmethodstothedistancematrices:
non-metricmultidimensionalscaling (NMDS) using the vegan(v2.6)
package’s metaMDS function117, and principal coordinate analysis
(PCoA)usingtheape(v5.6)package’spcoafunction118.Forboththe
mainpollencharacterdatasetanditssubsets,ourNMDSordinations
with k=2 yielded stress values below 0.2, effectively balancing the
preservationofthevariationstructureinourdatawithdimensionality
reductionforintuitivevisualisation.Wedidnotincludethecorrection
ofprincipalcoordinateaxes(PCos)inourdisparitypipeline,whichis
considerednottoaffectthestabilityoftheresults,butwouldresultin
asignificantreductioninthevarianceexplainedbythefirstfewPCos.

Characterisingdisparity
We used four key metrics related to size—Sum of Variances (SOV),
MeanPairwiseDistance(MPD),MeanDistancetoCentroid(MDC),and
Sum of Ranges (SOR)—to measure and describe the within-clade
morphologicaldisparitywithinourdataset.SOV,astheprimaryfocus,
alongside MPD, provides insights into the overall extent of the
exploredarea.MDChighlightshowdatapointsclusteraroundtheir
centrallocation,whereasSORexaminesthebreadthofthedistribution
within the morphospace. Given SOR’s sensitivity to samplesize, we
adjusted this metric using rarefaction prior to comparison. In our
rarefaction process, n species were randomly selected from each
groupwithoutreplacement,wherenequalsthenumberofspeciesin
thesmallestgroupminusone87.Toassessthereliabilityofordination-
basedmetrics,wealsocomparedMPDvaluesderivedfromtheraw
distance matrix—recommended for discrete character data by
Gerber119—withthosefromNMDSandPCoAordinations.

Tofacilitatecomparisonsofpollendisparitywithinangiosperms,
we adopted two informal grouping schemes (see Supplementary
Data1fordetails).Wetreatedangiospermsasasinglesubclass(Mag-
noliidae)withoutusingthesuperorderrankofChaseandReveal120,as
itcombinesafewextremelylargesuperorders(e.g.,Lilianae,Rosanae,
Asteranae)withmanysmallsuperorders,whichmaybelesssuitablefor
ourcomparativeanalyses.

G1.Angiospermsweredividedintosixmajorgrades/clades:the
ANA grade, magnoliids, Chloranthales, Ceratophyllales, monocots,
and eudicots (Fig. 1). The ANA grade comprises the three earliest
diverging angiosperm orders(Amborellales, Nymphaeales, and Aus-
trobaileyales). The subsequent five groups represent the five major
cladesofmesangiosperms.

G2.G2wasestablishedtofurthersubdividemonocotsandeudi-
cots, thus retaining the ANA grade, magnoliids, Chloranthales, and
Ceratophyllales.Formonocots,wesubdividedthemintocommelinids
andothermonocots.Withineudicots,weadoptedtheinformalclade
namessuperrosidsandsuperasteridsofAPGIV104:superrosidsinclude
fabids, malvids, and other superrosids (Saxifragales and Vitales),
whereas superasterids include campanulids, lamiids, and other
superasterids (Berberidopsidales, Caryophyllales, Cornales, Ericales,
andSantalales).Thefiveearly-divergingeudicotorders(Ranunculales,
Proteales,Trochodendrales,Buxales,andGunnerales),togetherwith
Dilleniales, were grouped as other eudicots. In total, G2 comprises
thirteengroups:ANAgrade(ANA),magnoliids(MAG),Chloranthales
(CHLO), Ceratophyllales (CER), commelinids (COMM), other mono-
cots(OthM),othereudicots(OthE),fabids(FAB),malvids(MAV),other
superrosids (OtSR), campanulids (CAM), lamiids (LAM), and other
superasterids (OtSA).

To decipher the significance of each axis in morphospace, we
aligned ordination axes with original variables using pseudo-factor
loadings.WeemployedK-meansclusteringtoexaminemorphospace
occupationandassessedthephylogeneticsignalusingBlomberg’sK121,
verifyingstatisticalsignificancethroughpermutationtestswith1000
iterations.Inaddition,weperformedaManteltestbetweenthepollen
morphologicaldistancematrixandthephylogeneticdistancematrix
(measuredinmillionsofyears)acrossallpairsofgenustips,inorderto
evaluatetheextenttowhichevolutionarytimecontributestodiver-
genceinpollenmorphology.Wealsoemployedasubgroupomission
approach to evaluate each subgroup’s contribution to the overall
disparity58.Specifically,werecalculatedthedisparityafterexcludinga
givensubgroupandthencomparedthisvaluetothetotaldisparity.
Theabsolutedifferencebetweenthesetwovaluesisproportionalto
thesubgroup’scontribution.

Theoreticalmorphospace
Beyondempiricaldata,weintroduced10,000theoreticalmorphotypes
toillustratepossiblecombinationsofangiospermpollenformsbasedon
the empirical pollen morphological matrix (Supplementary data 1).
These taxa were generated by randomly assembling observed states
across the 20 discrete traits described in Supplementary Table 2,
drawing on simulation strategies developed in earlier studies36,41. To
ensureinternalconsistencyandbiologicalplausibility,wepreservedthe
hierarchicalstructureofthedataset:ifaprimarycharacterwascodedas
missingorabsent,itsdependentsecondarycharacterswerealsocoded
as missing. The polymorphic coding used increases the number of
theoretical combinations, which we treated as missing data in the
simulations. The proportion of missing characters in our theoretical
matrix was adjusted to match that ofthe empirical dataset, ensuring
comparability between observed and theoretical morphotypes. The
resultingsimulateddatawasthenintegratedwiththeempiricaldatafor
distancematrixconstructionandsubsequentordinationanalyses.

Morphologicalconvergence
Wequantifiedmorphologicalconvergenceinangiospermpollenusing
theC1statisticofStayton122,whichmeasurestheproportionofmax-
imumancestraldivergencethathasbeenclosedbysubsequentevo-
lutionbetweentwolineages.WeemployedtheMORDdistancematrix
(alphavalueof0.5)andcalculatedC1forall3,530,641uniquegenus
pairs(=(1880tips–1)2)inourangiospermpollendataset.Genuspairs
with C1≥0.90 were considered highly convergent, and the lowest
sharedtaxonomicrankwasdeterminedforthesepairstoassessthe
taxonomicdistancebetweenconvergentlineages112,123.Itisimportant
tonotethataC1valueof1.0doesnotnecessarilyimplythatgeneraare
identicalacrossalltraits,butratherthattheirsubsequentevolution
has maximally reduced prior divergence since their most recent
commonancestor.Notably,insteadofadoptingthesuperorderrank,
weintroducedtwoinformaltaxonomicalranks(G1andG2)tocapture
themajorgroupstructureofangiosperms.

Ratetestofcharacterevolution
We analysed variations in rates of character evolution across the
phylogenetictreebyconductingdiscrete-characterratetestsusingthe
likelihood-ratiotestmethod,asoutlinedbyLloydetal.124.Thismethod
involves probability models that consider branch lengths and the
completenessofcharacterdatatocalculatedetailedratesofcharacter
change (λ). To enhance the statistical reliability of our results, we
employed the Benjamini and Hochberg false-discoveryrate method
withastringentsignificancethresholdof0.01.Thisstephelpsmanage
multiplecomparisons,bolsteringourconfidenceinthefindings.These
analyses were carried out for all 20 characters and three character
subsets,consistentlyusingCladdisver.0.63113.

Disparitythroughtime
Weemployedthe‘gradualsplits’modeldevelopedbyGuillermeand
Cooper125fortimeslicing,whichprobabilisticallyselectsancestorsor
descendantsforeachbootstrapreplicate,basedontheirrelativedis-
tancestodesignatedtimeslices.Thisapproachhelpssampledatafrom
NMDS and PCoA ordinations at various points in the evolutionary
timelineofangiosperms.Disparitywascalculatedatintervalsoffive
million years using 1000 bootstrapped replicates to define the dis-
parityestimatesandassociatedconfidenceintervals.Theanalysiswas
conducted under three scenarios representing different dating esti-
mates for the crown age of flowering plants: Cretaceous (139Ma),
Jurassic(187Ma),andTriassic(248Ma)crowns.Inaddition,tomitigate
potential sampling biases, we performed 100 random subsamples
(n=1000)ofthemorphologicalmatrixforDTTanalysis,focusingonly
onmonocotsandeudicotsduetotheirpredominanceinthedataset.
TheDTTresultsunderdifferentrarefactionlevelswerealsoexamined.

Correlations
Wecompiledspeciesdiversityinformationforallgeneraincludedinour
pollen disparity dataset from the Catalogue of Life126. Genome size
informationwasobtainedfromtheDNAC-valuesdatabase127,usingthe
average value per genus, yielding 770 genera of angiosperms repre-
senting57ordersand255families(SupplementaryData1).Pollination
typeswereobtainedfromStephensetal.128,with758generaoverlapping
withourdataset(SupplementaryData1).Afterexcludinggenerawith
polymorphicstates(treatedasmissingdata),thefinalcodingcomprised
708genera(62orders,403families)fortheabioticversusbioticcate-
gories and 659 genera (60 orders, 386 families) for the wind, water,
insect,andvertebratecategories.Althoughthecodingschemeusedby
Stephens et al.128 was primarily constructed at the species level, the
exclusion of polymorphic states in our analyses minimises potential
biases,andourfocusremainedexclusivelyonextantgenera.

Weobtainedpairwisedistancematricesforthemainpollendis-
parity dataset as well as subsets pertaining to each taxon pair. In
addition,wealsocalculatedpairwisedifferencesintaxonomicdiver-
sityandgenomesizeforeverytaxonpair.Consequently,wewereable
toevaluatethecorrelationbetweenthetwobyemployingphyloge-
netic Mantel tests asimplemented in the ‘EvolQG’R package129. We
additionally calculated lineages through time (LTT) to estimate the
temporalpatternsofgenusrichnessdepictedinthephylogenyusing
the‘ape’Rpackage(SupplementaryFig.16).Forpollinationtype,we
employedthePearson’scorrelationcoefficient(r)toassessthecor-
relation between the ordination axes of pollen disparity and the
categorical pollination coding, and we further conducted a PERMA-
NOVAtesttoexaminethemorphospaceoccupiedbydifferentpolli-
nationmodes117.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a fascinating and well-written paper. It provides a much-needed analysis of the phylogenetic pattern of pollen morphology and produces insights into the timing of angiosperm diversification. My expertise is in palynology and less so in phylogenetic methods, so my comments focus more on the morphological analysis than the phylogenetic work. That said, however, my main concern with the paper is the angiosperm phylogeny used in the study. The authors produce their own from published sequence data instead of relying on recently published phylogenies (e.g., Zuntini et al 2024). I do not believe that this will affect the interpretations or conclusions of the paper, given the broad patterns that this paper focuses on, but the authors should be aware that there will be a mismatch between the superorder clades that they discuss (Fabids, Malvids, Superasterids, Superrosids) and more recent delimitations. Given the speed at which genomic data is now being accumulated, and the potential for future reorganization of the angiosperm phylogeny, it would be helpful if the authors included a spreadsheet of the genera and orders they include within their larger named groups to help with transparency and interpretability of their analysis. I would also strongly encourage the authors to publish the list of genera in their analysis and their character scores as a second spreadsheet. This again would improve the transparency and interpretability of their analysis and encourage others to build on their work. Section-by-section points below: Abstract • “Endowed” is an odd word choice. Maybe simply “composed”? Introduction • Page 3, line 3: “synapomorphy OF equatorially tricolpate pollen” • “the processes of angiosperm pollen diversification”: This paper is more about pattern than process. Suggest that you emphasize the importance of documenting pattern to infer process. • “This gap hinders, and may even distort, our understanding of angiosperm radiation, particularly regarding deep evolutionary processes”: I agree with the sentiment, but it would be better if the authors made a more explicit statement of how this is a result of the previous sentence. Results • p 4: “a discrete-coded matrix”: Should note that 20 characters were coded, and the larger groupings (aperture, exine, shape) should be introduced and described/defined. Otherwise, the meaning of the shorthand terms used in the results (aperture, exine, shape) are hard to understand. • p 4: “The topology is congruent with recent phylogenetic frameworks 48-50”: Note that Zuntini et al 2024 does NOT have the same topology as previous phylogenies (e.g., APG IV) • p 5, paragraph 1: “plastome-based PPA tree”: Should include “the” before the word “plastome”. Also need to define what this is, particularly the acronym PPA. • p 5, paragraph 1: “a temporal pattern irreconcilable with current paleobotanical evidence”: It is not clear whether you are saying your results are irreconcilable or whether old tree scenarios are. Based on Coiro et al 2019 review of the Triassic fossil evidence, I would say that phylogenies that place the ancestral angiosperm node very early (Triassic or before) conflict most with the paleo record. • p 5, paragraph 2: “In our ordination analysis, we opted for Non-metric Multidimensional Scaling (NMDS) to process the pollen data”: process is not the correct word. Interpret? Analyze? • p 5: Pollen morphospace occupation (and elsewhere): all palynological terms should be defined when first introduced. When mentioning a trait (e.g., polarity), mention which larger grouping it belongs to (aperture, exine, shape). • p 5, last paragraph: “Compared to the theoretical morphospace, the empirical morphospace occupies approximately 43% of the area on the left side”: Language here is too casual and imprecise. Right vs left is arbitrary in a multivariate analysis. What is important is that only a portion of the theoretical morphospace is occupied. Instead of left and right, describe the axis by the traits with which it most strongly covaries, positively and negatively. • p 6, paragraph 1: “the highest levels”: What does this mean? • p 6, paragraph 1: “overall pollen disparity”: Is this within-clade or between clades? • p 7, paragraph 1: “overlap between the two lists”: What two lists? If this is a reference to Table S5, include the reference and explain the difference between the two sets of rankings. Discussion • p 9, paragraph 2: “These differences emphasize that varying scales and subjects of observation can yield different evolutionary insights.”: Is the argument that early pollen evolution was constrained? The wording is a little vague. • p 9, paragraph 3: “In animal taxa, basally-branching groups often exhibit higher levels of disparity than their more derived counterparts 15,24,25, a pattern also observed in the morphospace of angiosperm flowers 41,56.” This is repetitive, as the same point is made in the previous paragraph. • p 10: Synchronous Evolution of Pollen and Leaves: This section is speculative and has a tenuous connection to the rest of the paper. How does tricolpate pollen relate to hot wet environments? Recommend removing this section. • p 11, last paragraph: “while the Malpighiales are associated with the development of neotropical rainforests”: Need a citation for this. Malpighiales is a large and diverse order, but it is Malvales that is the more important Neotropical clade. Conclusions • Appreciate the mention of extinct diversity! Methods • p 15, paragraph 3: “with further subdivisions for detailed analysis”: As noted at the start of the review, you should specify that you are following APG IV clade deliminations, not Zuntini et al 2024. It is important that you list the orders and genera within each larger named group in a supplemental spreadsheet. • p 16, paragraph 1: More detail on how the theoretical morphospace was calculated would be helpful. Presumably these are random permutations of the pollen characters in Table S2 (which should be referenced here). Supporting information • Supplementary Table 2: A third column is needed to indicate which larger grouping each trait falls under (aperture, shape, exine). Happy to clarify any of the above comments. Surangi Punyasena (University of Illinois Urbana-Champaign)", "reviewer_2": "Dear Authors and Editor, I have reviewed the paper entitled \"Pulsed evolution shaped angiosperm pollen disparity\". I think it is a good study addressing the evolution of a very important plant character. On the one hand the authors scored several pollen traits to characterize pollen grains across a large set of species. On the author hand the authors generated a time calibrated molecular phylogeny of plants to look at the evolutionary history of these characters. By combining these two datasets and using comparative methods, they make a case for pulsed evolution of pollen. I think the paper is clearly written and the authors build on their work on this topic, namely the extensive series of papers on the “Evolution of Angiosperm Pollen”. A few questions I have are the following: Why a genus level pollen dataset? Are all the genera included monophyletic? Do the authors have any concern that if a genus in the dataset is not monophyletic their results would change? What happens when a genus is polymorphic for a trait? How does it get scored? What effect does it have to calculate disparity through time when the terminals in the tree are genera and not species? When assigning shapes to the pollen, one character involves \"Basic shape\" whereas a second \"Outline in polar view\". It is not clear to me how these are independant. Also, the character states of the second one includes as state 0 \"circular & elliptic\". Where would an elliptic pollen grain in polar view fall in the \"Basic shape\" variable? Looking at studies that review these characteristics of pollen, Walker and Doyle (1975) for example divide pollen shape into two basic types: globose and boat shaped. Does this equal globose-like vs filiform? Hesse et al. (2009) divide shape into oblate, spheroidal and prolate. So perhaps a little explanation of how some of those variables were established would be useful. Why were the traditional shape scoreing not used? Several old and recent studies have used morphometrics, from traditional approaches employing length to width rations, to geometric morphometric techniques such as elliptic Fourier analyses. Is there a reason why the authors chose not to use a quantitative approach to measure pollen grain shape and instead stuck to a discrete and simplified way of scoring pollen? The size variable has very unequal bin sizes. How were those size classes determined? Maybe I missed it but the pollen characters used are subdivided into three subtypes but I don't think it is clear which characters are assigned to each subgroup. Maybe in Supplementary table 2 the subgroups could be pointed out. Supplemental Information Page 1, paragraph 2: change \"Phylogenetic comparison methods\" to \"Phylogenetic comparative methods\" I appreciate that all the scripts, data and code are made available.", "reviewer_3": "This study applies an impressive morphological dataset of pollen characters and a time-calibrated phylogeny to document morphospace occupation and changes in morphological diversity through time and across major angiosperm groups. I found this paper well-written and interesting to read. There are several points that I would like to highlight: 1) the taxon sampling for extant species is exceptionally good and the overall amount of missing data is low, an outcome that clearly reflects considerable effort by the authors; 2) I have no specific criticisms regarding the methods, as the authors employed and compared different ordination methods (NMDS and PCoA) as well as various and disparity indices (SOR, SOV, MPD, MDC). In my opinion, this is an interesting paper as allow us comparative analysis of morphological variation in other angiosperms organs, such as flowers, leaves, and wood providing a more comprehensive understanding of the evolutionary radiation of angiosperms. Major comment The lack of fossil data. Luo et al. are clearly aware of the limitation of this study is that the estimation of pollen variation through time is based solely on extant taxa. I find no adequate justification for completely disregarding the valuable knowledge that has been compiled over decades by the paleobotanical community on the fossil pollen record. In my view, the fossil pollen record represents the only direct source of information for assessing morphological disparity in the deep past, and its analysis and quantification are essential for a comprehensive understanding of evolutionary patterns over time. More reliable macroevolutionary inferences require the inclusion of fossil taxa, as there are many examples in which the fossil record contain much more information about past evolutionary patterns than phylogenies, for example preserving unique character combinations that have not seen in living species and consequently cannot be reconstructed in ancestral state reconstructions. Incorporating such fossil data could significantly change the observed patterns of disparity through time. The authors indicate that earlier studies fossil pollen (e.g., Lupia, 1999; Doyle 1977) “were constrained by a limited understanding of evolutionary relationships among groups and were restricted in geographical and temporal extent”. I agree with this assessment; however, I believe that significant progress has been made in recent decades to improve our understanding of the fossil pollen record. For example, new species have been described (many with a great detail) and new localities have been sampled greatly expanding the temporal and geographical coverage of available data. I am not saying that the fossil record is perfect, fossil data are subject to several biases, but also inferring these patterns using the phylogenies of extant taxa is not accurate. This is my main concern; the approach considered in this study, while methodologically sound with its scope, may fall short of what is expected for a Nature Communications paper, especially compared to a recent article on a similar topic (Clark et al. 2023 Nat Plants), which integrated fossil and extant data to assess morphological disparity. One potential solution for this pitfall is that the authors should be explicit about the estimations based only on extant taxa, for example in the title or in the abstract. I think that a section comparing the fossil record (not only the patterns observed by Lupia or Doyle) with your results is missing. For instance, based on your findings, you indicate that rates of pollen evolution remained stable during the mid-Cretaceous and Paleogene. However, what does the pollen fossil record reveal about the Late Cretaceous? How did morphological diversification proceed during this interval? Was it similarly stable and low, or were there notable shifts in morphology? Additionally, which new morphological characters first appeared during this period? A discussion of these aspects would significantly enrich the interpretation of your results within a broader evolutionary and paleobotanical context. The main strength of this paper, in my view, is that the authors, given their taxon sampling, are able to offer a comprehensive comparison of disparity variation among extant angiosperm clades or lineages and provide a further discussion on this variation. For example: -why some extant clades appeared more restricted in the space than others? I see that the authors explained this topic in the section of “extant angiosperm pollen morphospace”, however, it would be interesting to know, for example: -the extensive morphospace occupation by fabids aligns with patterns observed in studies of floral or leaf morphospaces? -Why do monocots exhibit the highest morphological disparity despite not occupying the largest areas of the morphospace? -The ANA grade, Chloranthales, and the other superasterids groups have the lowest contributions to the overall disparity, however, to my knowledge those groups contributed relatively high to the overall floral disparity (see Lopez-Martinez et al 2024), why? -The comparison between pollen disparity and pollination mode was particularly interesting. The authors obtained the data of pollination mode from Stephens et al.; however, to my knowledge, the sampling might be substantially different. Stephens et al. included about 1200 extant taxa, with pollination mode mostly scored at species level, which contrasts with the sampling presented in this study. The authors should clarify these differences and explain how they account for the variation on pollination type within a Genera (e.g., Aquilegia shows vertebrate and insect pollination or even exhibit a mixed pollination strategies). Minor comments: The measure of disparity based on the sum of variances using the NMDS and PCoA ordination can be problematic when using discrete morphological characters. I think it is more accurate to measure disparity based on the distance matrix (please see Gerber, 2019. Use and misuse of discrete character data for morphospace and disparity analyses. Palaeontology 62: 305-319)."}, "author_response": "RESPONSE TO REVIEWERS´ COMMENTS\nNote: Line numbers refer to the revised manuscript with tracked changes\n(Revised_manuscript_tracked.docx).\nReviewer #1 (Remarks to the Author):\nThis is a fascinating and well-written paper. It provides a much-needed analysis of\nthe phylogenetic pattern of pollen morphology and produces insights into the timing\nof angiosperm diversification.\n>>> Thank you for your thorough review and positive insights. We appreciate all\nthe comments and have addressed all of them below.\nMy expertise is in palynology and less so in phylogenetic methods, so my\ncomments focus more on the morphological analysis than the phylogenetic work.\nThat said, however, my main concern with the paper is the angiosperm phylogeny\nused in the study. The authors produce their own from published sequence data\ninstead of relying on recently published phylogenies (e.g., Zuntini et al 2024). I do\nnot believe that this will affect the interpretations or conclusions of the paper, given\nthe broad patterns that this paper focuses on, but the authors should be aware that\nthere will be a mismatch between the superorder clades that they discuss (Fabids,\nMalvids, Superasterids, Superrosids) and more recent delimitations. Given the\nspeed at which genomic data is now being accumulated, and the potential for future\nreorganization of the angiosperm phylogeny, it would be helpful if the authors\nincluded a spreadsheet of the genera and orders they include within their larger\nnamed groups to help with transparency and interpretability of their analysis.\n>>> Thank you for your suggestion. We have listed the orders and major clade\naffiliations of all sampled angiosperm genera included in our study in Supplemental\nData 1, following both APG IV (2016) and Zuntini et al. (2024). We have also clearly\nstated this in the Methods section and highlight the differences between the two\nclassification systems, see lines 555-559.\nAbove the ordinal level, naming conventions within the APG system have remained\nfluid and largely informal. APG III (2009) recognized the need for names\ncorresponding to the major clades of angiosperms, given that the entire clade is\ntreated as a single subclass. In response, Chase and Reveal (2009) proposed a\nclassification with 18 superorders. However, APG IV did not adopt this system and\ninstead introduced the new informal terms Superasterids and Superrosids to assist\ncommunication and comprehension.\nIn our study, although the phylogenetic tree is based on both plastid and nuclear\ngenes, the recovered ordinal-level relationships are largely consistent with those\nrecognized by APG IV. Therefore, we adopted the informal clade names endorsed\nby the APG system. These names have become widely accepted within the\nbotanical community and serve a practical purpose in facilitating communication\nacross disciplines.\nZuntini et al. (2024) represents a major update to our understanding of angiosperm\nrelationships. Their study recovered 58 of the 64 currently accepted orders and\n406 of the 416 families as monophyletic and confirmed 85% of the inter-family\nrelationships reported in our Plastid Phylogenomic Angiosperm II (PPA II) project\n(Li et al., 2021).\nNotably, Zuntini et al. (2024) proposed a recircumscription of fabids and malvids,\nwhere the new fabid clade (Cucurbitales, Fabales, Fagales, and Rosales)\ncorresponds to what was previously defined as the nitrogen-fixing clade, while the\nformer COM clade (Celastrales–Oxalidales–Malpighiales) is now placed within\nmalvids. It is worth noting that APG IV had already acknowledged potential\ncytonuclear discordance within the COM clade based on earlier nuclear and\nmitochondrial data, and this uncertainty was explicitly marked in their ordinal-level\nchronograms.\nZuntini et al. (2024) also introduced adjustments to the placements of several\norders, particularly those previously unassigned to the four major monophyletic\nclades of core eudicots (campanulids, lamiids, fabids, and malvids), such as\nCaryophyllales, Santalales, Dilleniales, Gunnerales, Bruniales, and Icacinales. At\nthe same time, we note that definitions and interrelationships among major\nangiosperm groups—especially within the core eudicots—still vary across different\nversions of the Kew Tree of Life Explorer (https://treeoflife.kew.org/tree-of-life)\nassociated with the Zuntini et al. (2024) phylogeny.\nWe are confident that through continued international collaboration and\nconsensus-building—as reflected in the APG framework—these discrepancies will\ngradually be resolved, and a stable and broadly accepted classification will emerge.\nRefs cited in this Reply:\nAPG IV. An update of the Angiosperm Phylogeny Group classification for the\norders and families of flowering plants: APG IV. Bot J Linn Soc 181, 1-20 (2016).\nChase M. W., Reveal J. L. A phylogenetic classification of the land plants to\naccompany APG III. Bot J Linn Soc 161, 122-127 (2009).\nLi H.-T., et al. Plastid phylogenomic insights into relationships of all flowering plant\nfamilies. BMC Biol 19, 232 (2021).\nZuntini A. R., et al. Phylogenomics and the rise of the angiosperms. Nature 629,\n843-850 (2024).\nI would also strongly encourage the authors to publish the list of genera in their\nanalysis and their character scores as a second spreadsheet. This again would\nimprove the transparency and interpretability of their analysis and encourage\nothers to build on their work.\n2\n>>> We agree that transparency is crucial for reproducibility and further research.\nWe have included Supplemental data 1 containing the list of genera in our analysis\nand their character scores.\nSection-by-section points below:\nAbstract\n• “Endowed” is an odd word choice. Maybe simply “composed”?\n>>> We have changed “endowed with” to “enclosed within”, which we agree is\nmore accurate in describing the structural relationship between pollen grains and\ntheir sporopollenin walls.\nIntroduction\n• Page 3, line 3: “synapomorphy OF equatorially tricolpate pollen”\n>>> Revised.\n• “the processes of angiosperm pollen diversification”: This paper is more about\npattern than process. Suggest that you emphasize the importance of documenting\npattern to infer process.\n>>> We rephrased this as follows: “However, despite this importance, the large-\nscale patterns of angiosperm pollen diversification remain poorly quantified and\nunderstood. This lack of a quantitative framework limits—and may even mislead—\nour understanding of angiosperm radiation, particularly regarding deep\nevolutionary processes.”\n• “This gap hinders, and may even distort, our understanding of angiosperm\nradiation, particularly regarding deep evolutionary processes”: I agree with the\nsentiment, but it would be better if the authors made a more explicit statement of\nhow this is a result of the previous sentence.\n>>> See above.\nResults\n• p 4: “a discrete-coded matrix”: Should note that 20 characters were coded, and\nthe larger groupings (aperture, exine, shape) should be introduced and\ndescribed/defined. Otherwise, the meaning of the shorthand terms used in the\nresults (aperture, exine, shape) are hard to understand.\n>>> We have revised the related sentences (see lines 136-142) and clarified the\ncharacter definition and their groupings in supplemental Table S2 to improve\nreader comprehension.\n• p 4: “The topology is congruent with recent phylogenetic frameworks 48-50”:\nNote that Zuntini et al 2024 does NOT have the same topology as previous\nphylogenies (e.g., APG IV)\n3\n>>> We thank the reviewer for pointing this out. We agree that Zuntini et al. (2024)\ndiffers from earlier plastid-based phylogenies in terms of higher-level clade\ndefinitions (e.g., the recircumscription of fabids and malvids). We have revised this\nsentence to:\n\"The topology is broadly congruent with recent plastome-based phylogenetic\nframeworks (Li et al., 2019, 2021) at the ordinal level, although recent nuclear\nphylogenomic study (e.g., Zuntini et al., 2024) suggest alternative relationships,\nparticularly within the rosid clade. Our topology supports relationships\n[Chloranthales [magnoliids [monocots [Ceratophyllales + eudicots]]]] within the\nmesangiosperms.\"\nRefs cited in this Reply:\nLi H.-T., et al. Origin of angiosperms and the puzzle of the Jurassic gap. Nature\nPlants 5, 461-470 (2019).\nLi H.-T., et al. Plastid phylogenomic insights into relationships of all flowering plant\nfamilies. BMC Biol 19, 232 (2021).\nZuntini A. R., et al. Phylogenomics and the rise of the angiosperms. Nature 629,\n843-850 (2024).\n• p 5, paragraph 1: “plastome-based PPA tree”: Should include “the” before the\nword “plastome”. Also need to define what this is, particularly the acronym PPA.\n>>> We changed it to “the plastid phylogenomic angiosperm (PPA) tree”.\n• p 5, paragraph 1: “a temporal pattern irreconcilable with current paleobotanical\nevidence”: It is not clear whether you are saying your results are irreconcilable or\nwhether old tree scenarios are. Based on Coiro et al 2019 review of the Triassic\nfossil evidence, I would say that phylogenies that place the ancestral angiosperm\nnode very early (Triassic or before) conflict most with the paleo record.\n>>> Thank you for this helpful clarification. Our intention was to convey that the\n“old tree” scenarios, which place the crown age of many major angiosperm\nlineages in the Triassic, are incongruent with current paleobotanical evidence. To\naddress this, we have revised the sentence for clarity:\n> “Notably, our dated trees align more closely with the ‘young tree’ scenario at\nordinal and familial levels, in contrast to ‘old tree’ scenarios that place most ordinal\ndiversifications in the Triassic—a timeframe that conflicts with current\npaleobotanical evidence…”\n• p 5, paragraph 2: “In our ordination analysis, we opted for Non-metric\nMultidimensional Scaling (NMDS) to process the pollen data”: process is not the\ncorrect word. Interpret? Analyze?\n>>> We have changed “process” to “analyze”.\n4\n• p 5: Pollen morphospace occupation (and elsewhere): all palynological terms\nshould be defined when first introduced. When mentioning a trait (e.g., polarity),\nmention which larger grouping it belongs to (aperture, exine, shape).\n>>> We have added a clear reference to Supplementary Table S2 at the start of\nthe results to define all palynological characters and their groupings. We also\nspecify the morphological category (shape, aperture, or exine) for each trait at first\nmention (e.g., polarity (shape)) throughout the main text.\n• p 5, last paragraph: “Compared to the theoretical morphospace, the empirical\nmorphospace occupies approximately 43% of the area on the left side”: Language\nhere is too casual and imprecise. Right vs left is arbitrary in a multivariate analysis.\nWhat is important is that only a portion of the theoretical morphospace is occupied.\nInstead of left and right, describe the axis by the traits with which it most strongly\ncovaries, positively and negatively.\n>>> We have rephrased the sentence quoted above as follows: “Compared to the\ntheoretical morphospace, the empirical morphospace occupies approximately 43%\nof the total area (Fig. 2). This occupied region clusters toward the negative end of\nNMDS Axis 1, which covaries most strongly with traits such as pollen size and the\npresence/absence of tectum. Within this empirical space, 95% of extant genera\nare concentrated in approximately 32% of the area (Fig. 2).”\n• p 6, paragraph 1: “the highest levels”: What does this mean?\n>>> We have revised the phrase “the highest levels” to “the highest disparity levels”\nin the relevant section to improve clarity and precision.\n• p 6, paragraph 1: “overall pollen disparity”: Is this within-clade or between\nclades?\n>>> We added “For the within-clade analysis, …” at the beginning of the paragraph,\nand we have also emphasized this point in the Methods section.\n• p 7, paragraph 1: “overlap between the two lists”: What two lists? If this is a\nreference to Table S5, include the reference and explain the difference between\nthe two sets of rankings.\n>>> We apologize for the lack of clarity in our citation. In this section, we were\nspecifically referring to the information presented in Figure 3. We have revised the\nwording accordingly (lines 237–241) to ensure the reference is explicit, and we\nhave updated Supplementary Table S3 to include the rates of morphological\nevolution for aperture traits, which were previously omitted.\nDiscussion\n• p 9, paragraph 2: “These differences emphasize that varying scales and subjects\nof observation can yield different evolutionary insights.”: Is the argument that early\npollen evolution was constrained? The wording is a little vague.\n5\n>>> We agree that the two consecutive uses of “These differences” may have\ncaused confusion. To improve clarity, we have reworded the sentence as:\n“Moreover, reproductive cells fortified with sporopollenin have been found\nthroughout land plant history 54, and the emergence of sporopollenin is considered\na key adaptation to terrestrial life 55. Flowers, by contrast, are a more recent\ninnovation. Taken together, these contrasting patterns of disparity reflect how\nevolutionary insights are shaped by temporal and taxonomic scales, as well as the\nfocus of observation.”.\n• p 9, paragraph 3: “In animal taxa, basally-branching groups often exhibit higher\nlevels of disparity than their more derived counterparts 15,24,25, a pattern also\nobserved in the morphospace of angiosperm flowers 41,56.” This is repetitive, as\nthe same point is made in the previous paragraph.\n>>> We would like to clarify that the two statements refer to conceptually distinct\naspects of disparity. The first addresses temporal patterns, contrasting the\ncommonly held early maximal disparity model with our findings of episodic\nincreases in pollen disparity. The second focuses on phylogenetic patterns,\nspecifically the assumption that basally branching clades tend to be more diverse\nthan derived ones. To improve clarity, we have revised the phrasing slightly.\n• p 10: Synchronous Evolution of Pollen and Leaves: This section is speculative\nand has a tenuous connection to the rest of the paper. How does tricolpate pollen\nrelate to hot wet environments? Recommend removing this section.\n>>> We agree that this section is not directly integrated with the core focus of the\nmanuscript. In response, we have removed the section on synchronous evolution\nof pollen and leaves. Some of the ideas previously discussed in that section have\nnow been integrated into the “Two Main Pulses in Angiosperm Pollen Disparity”\npart of the Discussion to ensure better alignment with the manuscript’s central\nnarrative see lines 352-360.\n• p 11, last paragraph: “while the Malpighiales are associated with the\ndevelopment of neotropical rainforests”: Need a citation for this. Malpighiales is a\nlarge and diverse order, but it is Malvales that is the more important Neotropical\nclade.\n>>> We agree that Malvales is a large and important Neotropical order. However,\nseveral studies have highlighted the dominant presence of Malpighiales in lowland\ntropical rainforests, especially in the woody understory layers. For instance,\nDavis et al. (2005) noted that Malpighiales constitute up to 28% of species and 38% of\nstems in such environments. Furthermore, in Amazonian forests, Malpighiales\ninclude 10 families contributing 43 of the 227 species that make up half of all trees\n≥10 cm diameter at breast height (dbh), representing ~20% of this dominant tree\nsubset (ter Steege et al. 2013). In combination with Ericales, Malpighiales account\nfor more than half of the understory stems in tropical rainforests in Asia and Africa\n(Davis et al. 2005). We revised the sentence to read: “the Malpighiales and Ericales\n6\nare strongly represented in lowland tropical rainforests and have been associated\nwith the development of Neotropical rainforests 81,82”, and added citations to\nsupport this statement.\nRefs cited in this Reply:\nDavis, C.C., Webb, C.O., Wurdack, K.J., Jaramillo, C.A. & Donoghue, M.J.\nExplosive radiation of Malpighiales supports a mid-Cretaceous origin of modern\ntropical rain forests. The American Naturalist, 165:3, E36-E65 (2005).\nter Steege, H. et al. Hyperdominance in the Amazonian tree flora. Science,\n342(6156), 1243092. (2013)\nConclusions\n• Appreciate the mention of extinct diversity!\n>>> We appreciate your recognition.\nMethods\n• p 15, paragraph 3: “with further subdivisions for detailed analysis”: As noted at\nthe start of the review, you should specify that you are following APG IV clade\ndeliminations, not Zuntini et al 2024. It is important that you list the orders and\ngenera within each larger named group in a supplemental spreadsheet.\n>>> We have now added this information to Supplemental Data 1.\n• p 16, paragraph 1: More detail on how the theoretical morphospace was\ncalculated would be helpful. Presumably these are random permutations of the\npollen characters in Table S2 (which should be referenced here).\n>>> Thank you for this suggestion. We have now added detailed information in the\nMethods section (lines 648-659) describing how the theoretical morphospace was\nconstructed. Specifically, the 10,000 theoretical morphotypes were generated by\nrandomizing observed character states for the 20 discrete-coded traits listed in\nSupplementary Table S2. We also enforced empirical levels of missing data by trait\nto ensure comparability. These simulated data were then included alongside\nempirical taxa for ordination and disparity analyses.\nSupporting information\n• Supplementary Table 2: A third column is needed to indicate which larger\ngrouping each trait falls under (aperture, shape, exine).\n>>> Revised.\nHappy to clarify any of the above comments.\n>>> Thank you very much for your constructive comments and kind offer to clarify.\nWe have carefully addressed all points and are happy to follow up if further\nclarification is needed.\nSurangi Punyasena (University of Illinois Urbana-Champaign)\n7\n## Reviewer #2 (Remarks to the Author)\nDear Authors and Editor,\nI have reviewed the paper entitled \"Pulsed evolution shaped angiosperm pollen\ndisparity\". I think it is a good study addressing the evolution of a very important\nplant character. On the one hand the authors scored several pollen traits to\ncharacterize pollen grains across a large set of species. On the author hand the\nauthors generated a time calibrated molecular phylogeny of plants to look at the\nevolutionary history of these characters. By combining these two datasets and\nusing comparative methods, they make a case for pulsed evolution of pollen. I think\nthe paper is clearly written and the authors build on their work on this topic, namely\nthe extensive series of papers on the “Evolution of Angiosperm Pollen”.\n>>> We sincerely thank the reviewer for the positive and encouraging feedback.\nA few questions I have are the following: Why a genus level pollen dataset?\nAre all the genera included monophyletic? Do the authors have any concern that\nif a genus in the dataset is not monophyletic their results would change? What\nhappens when a genus is polymorphic for a trait? How does it get scored? What\neffect does it have to calculate disparity through time when the terminals in the tree\nare genera and not species?\n>>> We thank the reviewer for these thoughtful and important questions regarding\nour use of a genus-level pollen dataset. Below we respond to each point in turn:\n(1) Why use genera as terminal units?\nWe chose the genus level for several reasons. First, genus-level sampling is widely\nused in macroevolutionary studies as a way to reduce species-level sampling\nbiases while still capturing meaningful proxies for ecological and morphological\ndiversity (Jablonski 2017). Notably, Linnaean genera continue to be treated as\nbiologically meaningful units, and the evolutionary reality of both species and\ngenera has been extensively discussed (e.g., Barraclough and Humphreys, 2015).\nAmong higher taxa, genera often exhibit the highest degree of evolutionary\ncoherence or “shared fate”, reflected in shared evolutionary trajectories and\npredictable morphological patterns among constituent species.\nSecond, evolutionary signals tend to vary by taxonomic rank: higher-level clades\noften represent the emergence of new body plans, while lower-level clades capture\nthe dynamics of species formation and turnover (Valentine 2004; Wagner 2014;\nJablonski 2017). Since our focus is on large-scale evolutionary patterns in pollen\nmorphology, intra-generic evolutionary history lies beyond the scope of this study.\nThird, the genus level has long been the primary taxonomic unit in palynology, both\nfor extant and fossil taxa, largely because of the morphological homogeneity of\npollen among species within genera. Pollen morphology is traditionally described\nat the genus level, resulting in a wealth of comparative data at that rank. Although\nspecies-level datasets would be ideal, the current state of taxonomic and\n8\nmorphological coverage makes genus-level sampling both more feasible and more\ncomprehensive.\n(2) Are all included genera monophyletic, and what is the effect of using genera\nrather than species as terminals?\nThis is closely related to the previous point. Although genera are not perfect units,\nthey typically correspond well to genealogical groups and can effectively serve as\nproxies for low-level clades of closely related species (Jablonski 2017). In our study,\nwe carefully checked the taxonomic status of each genus using global databases\nsuch as the Catalogue of Life (COL) and Plants of the World Online (POWO).\nWhen building the phylogeny, we excluded taxa with uncertain placements to\nensure the monophyly of accepted genera, families, and orders.\nMost currently accepted angiosperm genera are now well circumscribed and are\ngenerally supported as monophyletic by available molecular data, even in large-\nscale supermatrix analyses with incomplete sampling (e.g., Zanne et al. 2014;\nSmith et al. 2018). In addition, our sampling strategy emphasized broad\nphylogenetic representativeness, including all major angiosperm orders and most\nmajor intrafamilial clades, ensuring that deeper branching events leading to extant\nlineages were well captured.\nTo evaluate the stability of our disparity-through-time analyses, we also performed\n100 replicate analyses based on random subsampling of 1000 genera from our\ndataset. These analyses consistently recovered similar temporal patterns,\nincluding pulses of morphological disparity, indicating that our key results are\nrobust to variation in taxon sampling and are unlikely to be artifacts of genus-level\nnon-monophyly.\n(3) How is polymorphism within genera handled in character scoring?\nTo represent known within-genus variation, we adopted a polymorphic coding\nstrategy that retains all observed states without modification (e.g., scoring ‘0&1&2’).\nThis remains one of the most commonly used methods for scoring polymorphisms\nin discrete characters (Wortley et al. 2015; Watanabe 2016), especially when\ncharacter state frequencies are not well known. While frequency-based coding\nmay in principle offer greater resolution, it is infeasible for our dataset due to the\nlimited availability of species-level pollen data for many genera. Subsequent\nanalysis workflow for calculating pairwise distances (MORD distance metric) can\nhandle polymorphisms.\nAlternative approaches such as “democratic coding” (scoring only the most\ncommon state) have been used in some of our prior work, but we chose not to\napply this here because it introduces subjective assumptions (e.g., equating\nfrequency with ancestral state). Given current data limitations, we believe that\nretaining observed polymorphisms is a more transparent and biologically honest\napproach.\nOverall, our focus on discrete, basic traits of pollen morphology, coupled with a\nbroad macroevolutionary scope targeting deep divergences in angiosperm history,\nsupports the use of genus-level units and polymorphic coding. We have further\n9\nclarified our rationale for this approach in the Methods (lines 537-551, 580-595)\nand Supplementary Methods sections.\nRefs cited in this Reply:\nJablonski D. Approaches to Macroevolution: 1. General Concepts and Origin of\nVariation. Evolutionary Biology 44, 427-450 (2017).\nSmith, S. A. & Brown, J. W. Constructing a broadly inclusive seed plant phylogeny.\nAm. J. Bot. 105, 302–314 (2018).\nValentine, J. W. On the origin of phyla. Chicago: University of Chicago Press\n(2004).\nWagner P. J., Estabrook G. F. Trait-based diversification shifts reflect differential\nextinction among fossil taxa. Proc Natl Acad Sci U S A 111, 16419-16424 (2014).\nWatanabe, A. The impact of poor sampling of polymorphism on cladistic analysis.\nCladistics, 32: 317-334 (2016).\nWortley A. H., Wang H., Lu L., Li D.-Z., Blackmore S. Evolution of angiosperm\npollen. 1. introduction. Ann Mo Bot Gard 100, 177-226 (2015).\nZanne, A. E. et al. Three keys to the radiation of angiosperms into freezing\nenvironments. Nature 506, 89–92 (2014).\nWhen assigning shapes to the pollen, one character involves \"Basic shape\"\nwhereas a second \"Outline in polar view\". It is not clear to me how these are\nindependant. Also, the character states of the second one includes as state 0\n\"circular & elliptic\". Where would an elliptic pollen grain in polar view fall in the\n\"Basic shape\" variable?\n>>> We appreciate the reviewer’s careful attention to our character definitions,\nand we acknowledge that additional clarification is warranted. We have now added\na more detailed explanation to the Supplementary Methods, and provide a\nsummary here:\nWortley et al. (2015), the comprehensive overview of the \"Evolution of Angiosperm\nPollen\" series, outlines our character selection and coding approach. The study\nbuilds upon classic palynological literature and breaks down complex or composite\nterms into their most fundamental components.\nFollowing Walker and Doyle (1975), angiosperm pollen shape can be broadly\ncategorized as either nonfixiform (without a definite shape, e.g., filiform grains) or\nfixiform (with a definite shape). The latter can be further divided into two major\nshape classes: boat-shaped and globose. Wortley et al. (2015) adopted this\nframework. However, “basic shape” alone is insufficient to describe pollen\nmorphology comprehensively. It has thus become standard to describe shape\n10\nusing two additional traits—equatorial and polar views. In equatorial view, grains\nare categorized by shape class, defined by the ratio of the polar to equatorial axes\n(P/E). In polar view, outlines are described using more specific terms such as\ncircular, elliptic, polygonal, or lobate. Together, these three descriptors—basic\nshape, shape class, and outline in polar view—offer a composite description of the\n3D shape of pollen grains. It is important to note that filiform pollen represents a\nspecial case for which most shape descriptors become inapplicable. For such\ngrains, only basic shape is scored, and equatorial or polar outlines are coded as\n“not applicable”.\nIn practice, pollen shape is often influenced by the harmomegathic effect (also\nknown as the Wodehouse effect), especially in herbarium or fossil material\n(Halbritter and Hesse, 2004). This phenomenon can cause pollen grains to\ncollapse in varied and unpredictable ways, significantly altering their apparent\nmorphology. In our dataset, seven characters describe grain shape (characters 1–\n7), including basic shape, outline in polar view, and size, all of which can be\naffected to some degree by harmomegathy.\nBecause our study integrates data from diverse sources—each capturing pollen\ngrains at different hydration states—we adopted a harmonized coding strategy. For\nthe basic shape character, we follow the conceptual distinction between\nnonfixiform and fixiform pollen. For boat-shaped grains, we note that although Punt\net al. (2007) did not define this state, Halbritter et al. (2018) described it as “a\ncharacteristic shape of sulcate pollen grains in dry condition as a consequence of\nharmomegathy.” While this term can be useful in paleopalynology, particularly for\nidentifying monosulcate grains under light microscopy, we generally excluded it\nfrom disparity analyses due to its high plasticity. Recent studies of pollen\nmechanics and geometry have shown that transitions from globose to boat-shaped\nforms under desiccation are strongly influenced by aperture architecture (Katifori\net al. 2010; Božič et al. 2019).\nThe shape class character, likewise influenced by hydration state, is typically\ninapplicable to boat-shaped grains. Wortley et al. (2015) found this trait to be highly\nhomoplastic and relatively uninformative for phylogenetic inference. In our initial\nscoring, this character was missing in over 70% of genera—far exceeding our 25%\nthreshold for reliable inclusion. For these reasons, we excluded shape class from\nall disparity and morphospace analyses.\nWe hope this additional clarification addresses the reviewer’s concern, and we\nhave updated the Supplementary Methods to reflect this explanation in full.\nRefs cited in this Reply:\nBožič A., Šiber A. Mechanical design of apertures and the infolding of pollen grain.\nProc Natl Acad Sci U S A 117, 26600-26607 (2020).\nHalbritter H., Hesse M. Principal modes of infoldings in tricolp(or)ate Angiosperm\npollen. Grana 43, 1-14 (2004).\nSvojtka M., Frosch-Radivo A. Illustrated Pollen Terminology. Springer: Cham,\nSwitzerland (2018).\nKatifori E., Alben S., Cerda E., Nelson D. R., Dumais J. Foldable structures and\nthe natural design of pollen grains. Proc Natl Acad Sci U S A 107, 7635-7639\n(2010).\nPunt W., Hoen P. P., Blackmore S., Nilsson S., Le Thomas A. Glossary of pollen\nand spore terminology. Rev Palaeobot Palynol 143, 1-81 (2007).\nWalker J. W., Doyle J. A. The Bases of Angiosperm Phylogeny: Palynology. Ann\nMo Bot Gard 62, 664-723 (1975).\nWortley A. H., Wang H., Lu L., Li D.-Z., Blackmore S. Evolution of angiosperm\npollen. 1. introduction. Ann Mo Bot Gard 100, 177-226 (2015).\nLooking at studies that review these characteristics of pollen, Walker and Doyle\n(1975) for example divide pollen shape into two basic types: globose and boat\nshaped. Does this equal globose-like vs filiform? Hesse et al. (2009) divide shape\ninto oblate, spheroidal and prolate. So perhaps a little explanation of how some of\nthose variables were established would be useful. Why were the traditional shape\nscoreing not used?\n>>> See above.\nSeveral old and recent studies have used morphometrics, from traditional\napproaches employing length to width rations, to geometric morphometric\ntechniques such as elliptic Fourier analyses. Is there a reason why the authors\nchose not to use a quantitative approach to measure pollen grain shape and\ninstead stuck to a discrete and simplified way of scoring pollen?\n>>> We thank the reviewer for raising this point regarding the choice of discrete\ncharacter scoring over quantitative morphometric approaches.\nWhile classical and geometric morphometrics—such as length-to-width ratios and\nelliptic Fourier analyses—are powerful tools, they are most effective when applied\nwithin relatively homogeneous morphospaces, where the forms under study show\nmoderate variation and conserved outlines. However, their utility diminishes when\nmorphological disparity becomes extensive, as is the case in our broad survey\nacross all major angiosperm lineages.\nAs noted by Oyston et al. (2016), classical morphometric approaches \"are most\nsuitable when there is reasonable conservation of form, but lose traction where\nmorphological differences become greater (e.g., in comparisons across higher\ntaxa).\" Discrete character matrices, in contrast, provide a practical and scalable\nmeans of comparing a much wider range of morphologies, especially when dealing\nwith complex biological structures such as pollen.\nmicrostructural features—many of which cannot be reliably captured by size ratios\nor outline shape alone. While discrete coding may offer less geometric precision,\nit allows for the inclusion of key features (e.g., aperture type, exine structure,\npolarity) that are biologically and phylogenetically informative, but difficult to reduce\nto continuous metrics. In this sense, the flexibility of discrete coding—despite its\ncoarser granularity—remains advantageous for capturing the full extent of disparity\nin large-scale evolutionary analyses.\nIn our study, we implemented a carefully designed and standardized character\nmatrix (see Supplementary Table S2), following long-standing palynological\ntraditions and prior morphospace-based frameworks. We also added a sentence\nin the Methods section to briefly address this issue (lines 582-590). While we\nacknowledge the value of morphometric methods and see potential for their\ncomplementary use in more focused clade-level studies, our approach was\ndesigned to maximize comparability and coverage across the whole angiosperm\npollen.\nRefs cited in this Reply:\nOyston J. W., Hughes M., Gerber S., Wills M. A. Why should we investigate the\nmorphological disparity of plant clades? Ann Bot 117, 859-879 (2016).\nThe size variable has very unequal bin sizes. How were those size classes\ndetermined?\n>>> The pollen size classes used in our dataset follow the convention of Erdtman\n(1952) and Walker and Doyle (1975). Pollen size is known to vary depending on\nhydration status and specimen preparation method, and may also exhibit natural\nintraspecific variation. To accommodate these sources of variability, size classes\nhave traditionally been defined using broad bins that reflect approximate orders of\nmagnitude (see Walker and Doyle 1975; Wortley et al. 2015; Halbritter et al. 2018;\nPalDat database). We adopted the same categories: very small (<10 μm), small\n(10–24 μm), medium (25–49 μm), large (50–99 μm), very large (100–199 μm), and\ngigantic (>200 μm).\nWe have further clarified this in the Supplementary Methods sections.\nRefs cited in this Reply:\nErdtman G. Pollen morphology and plant taxonomy: Angiosperms. Almqvist and\nWiksell (1952).\nHalbritter H., Ulrich S., Grímsson F., Weber M., Zetter R., Hesse M., Buchner R.,\nSvojtka M., Frosch-Radivo A. Illustrated Pollen Terminology. Springer: Cham,\nSwitzerland (2018).\nWalker J. W., Doyle J. A. The Bases of Angiosperm Phylogeny: Palynology. Ann\nMo Bot Gard 62, 664-723 (1975).\npollen. 1. introduction. Ann Mo Bot Gard 100, 177-226 (2015).\nHalbritter H, Ulrich S, Grímsson F, et al. Illustrated pollen terminology[M]. Springer,\n2018.\nMaybe I missed it but the pollen characters used are subdivided into three subtypes\nbut I don't think it is clear which characters are assigned to each subgroup. Maybe\nin Supplementary table 2 the subgroups could be pointed out.\n>>> We have clarified the character definition and their groupings in supplemental\nTable S2 to improve reader comprehension.\nSupplemental Information\nPage 1, paragraph 2: change \"Phylogenetic comparison methods\" to \"Phylogenetic\ncomparative methods\"\n>>> Revised.\nI appreciate that all the scripts, data and code are made available.\n>>> Thank you—we appreciate the reviewer’s recognition of our efforts.\nReviewer #3 (Remarks to the Author):\nThis study applies an impressive morphological dataset of pollen characters\nand a time-calibrated phylogeny to document morphospace occupation and\nchanges in morphological diversity through time and across major angiosperm\ngroups. I found this paper well-written and interesting to read. There are several\npoints that I would like to highlight: 1) the taxon sampling for extant species is\nexceptionally good and the overall amount of missing data is low, an outcome\nthat clearly reflects considerable effort by the authors; 2) I have no specific\ncriticisms regarding the methods, as the authors employed and compared\ndifferent ordination methods (NMDS and PCoA) as well as various and disparity\nindices (SOR, SOV, MPD, MDC).\nIn my opinion, this is an interesting paper as allow us comparative analysis of\nmorphological variation in other angiosperms organs, such as flowers, leaves,\nand wood providing a more comprehensive understanding of the evolutionary\nradiation of angiosperms.\n>>> We sincerely thank the reviewer for the positive evaluation of our study and\ntheir recognition of our efforts in assembling a broad taxonomic sampling with\nminimal missing data.\nMajor comment\nis that the estimation of pollen variation through time is based solely on extant\ntaxa. I find no adequate justification for completely disregarding the valuable\nknowledge that has been compiled over decades by the paleobotanical\ncommunity on the fossil pollen record. In my view, the fossil pollen record\nrepresents the only direct source of information for assessing morphological\ndisparity in the deep past, and its analysis and quantification are essential for a\ncomprehensive understanding of evolutionary patterns over time. More reliable\nmacroevolutionary inferences require the inclusion of fossil taxa, as there are\nmany examples in which the fossil record contain much more information about\npast evolutionary patterns than phylogenies, for example preserving unique\ncharacter combinations that have not seen in living species and consequently\ncannot be reconstructed in ancestral state reconstructions. Incorporating such\nfossil data could significantly change the observed patterns of disparity through\ntime.\nThe authors indicate that earlier studies fossil pollen (e.g., Lupia, 1999; Doyle\n1977) “were constrained by a limited understanding of evolutionary\nrelationships among groups and were restricted in geographical and temporal\nextent”. I agree with this assessment; however, I believe that significant\nprogress has been made in recent decades to improve our understanding of\nthe fossil pollen record. For example, new species have been described (many\nwith a great detail) and new localities have been sampled greatly expanding\nthe temporal and geographical coverage of available data. I am not saying that\nthe fossil record is perfect, fossil data are subject to several biases, but also\ninferring these patterns using the phylogenies of extant taxa is not accurate.\nThis is my main concern; the approach considered in this study, while\nmethodologically sound with its scope, may fall short of what is expected for a\nNature Communications paper, especially compared to a recent article on a\nsimilar topic (Clark et al. 2023 Nat Plants), which integrated fossil and extant\ndata to assess morphological disparity. One potential solution for this pitfall is\nthat the authors should be explicit about the estimations based only on extant\ntaxa, for example in the title or in the abstract.\nI think that a section comparing the fossil record (not only the patterns observed\nby Lupia or Doyle) with your results is missing. For instance, based on your\nfindings, you indicate that rates of pollen evolution remained stable during the\nmid-Cretaceous and Paleogene. However, what does the pollen fossil record\nreveal about the Late Cretaceous? How did morphological diversification\nproceed during this interval? Was it similarly stable and low, or were there\nnotable shifts in morphology? Additionally, which new morphological characters\nfirst appeared during this period? A discussion of these aspects would\nsignificantly enrich the interpretation of your results within a broader\nevolutionary and paleobotanical context.\nThe main strength of this paper, in my view, is that the authors, given their taxon\nsampling, are able to offer a comprehensive comparison of disparity variation\n15\namong extant angiosperm clades or lineages and provide a further discussion\non this variation.\n>>> We fully agree that the fossil pollen record offers invaluable insights into past\nmorphological disparity and long-term evolutionary dynamics, and acknowledge\nthat significant progress has been made in recent decades in paleopalynology. We\nhave added additional text to the discussion (section ‘Two Main Pulses in\nAngiosperm Pollen Disparity’) that links our findings, and particularly the increases\nin disparity during the mid-Cretaceous and Paleogene, to the fossil pollen record.\nWe have also updated the title and added explicit statements in both the Abstract\nand Discussion noting that our analyses are based solely on extant taxa.\nIntegrating the morphological diversity of both extant and fossil pollen across the\nwhole angiosperms remains an extraordinarily challenging task. As noted in the\nintroduction to the paper, our aim in this study was to work in an explicit\nphylogenetic context to allow us to analyse both disparity through time and rates\nof morphological evolution. The plant fossil record, however, is mostly comprised\nof allochthonous and fragmentary remains (Cleal et al. 2021) that are challenging\nto meaningfully include in phylogenetic analyses. Even rigorously evaluating the\ntaxonomic affinities (e.g., to orders or families) of early angiosperm pollen types\nremains challenging, with many pre-Neogene palynotaxa having uncertain\nbotanical affiliations (Cleal et al. 2021; Friis et al. 2024). Consequently, large-scale\nsyntheses of the angiosperm fossil record often omit pollen from their datasets\n(e.g., Silvestro et al. 2021) —a marked contrast with the rich, regionally detailed\npalynostratigraphic studies.\nIncorporating large pollen fossil datasets—collected across widely varying\ngeographical and temporal scales—into an extant-focused phylogenetic\nframework without introducing bias is therefore beyond the scope of this study,\nalthough this is an important direction for future work.\nWe also note that the recent study by Clark et al. (2023), which incorporated fossil\ndata to analyze disparity across the entire plant kingdom, relied on a limited subset\nof assignable fossils and did not report any substantial changes in the overall\nmacroevolutionary patterns resulting from their inclusion. Their broader\nphylogenetic scope have allowed more reliable assignment of plant macrofossil to\nhigher-level clades (e.g., class).\nUltimately, paleontologists and neontologists investigate the same tree of life, and\na comprehensive understanding of macroevolution must be informed by both\nphylogenetic inference from living lineages and direct evidence from the fossil\nrecord.\nRefs cited in this Reply:\nClark J. W., et al. Evolution of phenotypic disparity in the plant kingdom. Nature\nPlants 9, 1618-1626 (2023).\nCleal C., et al. Palaeobotanical experiences of plant diversity in deep time. 1: How\nwell can we identify past plant diversity in the fossil record? Palaeogeogr,\nFriis E. M., Crane P. R., Pedersen K. R. The Cretaceous diversification of\nangiosperms: perspectives from mesofossils. Geological Society, London, Special\nPublications 544, 393-432 (2024).\nSilvestro D., et al. Fossil data support a pre-Cretaceous origin of flowering plants.\nNature Ecology & Evolution 5, 449-457 (2021).\nFor example:\n-why some extant clades appeared more restricted in the space than others? I\nsee that the authors explained this topic in the section of “extant angiosperm\npollen morphospace”, however, it would be interesting to know, for example:\n-the extensive morphospace occupation by fabids aligns with patterns observed\nin studies of floral or leaf morphospaces?\n>>> Within eudicots, López-Martínez et al. (2024) showed that floral disparity in\nfabids, the other superrosids grade, and the other superasterids grade was\ncomparable, though all were lower than in the other eudicots grade. In contrast,\nour results on pollen disparity indicate that fabids exhibit the second highest\ndisparity level, only after the other superasterids grade. This highlights that\ndifferent morphological systems (floral vs. pollen traits) can reveal distinct aspects\nof morphological diversification.\nTo address the reviewers’ comments regarding morphospace comparisons, we\nhave moved the section “Extant Angiosperm Pollen Morphospace” to the beginning\nof the Discussion and expanded its content to provide a more comprehensive\nexplanation of pollen morphospace structure, see lines 287-321.\nRefs cited in this Reply:\nLópez-Martínez A. M., Magallón S., von Balthazar M., Schönenberger J., Sauquet\nH., Chartier M. Angiosperm flowers reached their highest morphological diversity\nearly in their evolutionary history. New Phytol 241, 1348-1360 (2024).\n-Why do monocots exhibit the highest morphological disparity despite not\noccupying the largest areas of the morphospace?\n>>> Monocots show the highest values in three indices (SOV, MPD, MDC), which\nreflect internal spread, while not occupying the largest morphospace area (as\nmeasured by SOR), which reflects the overall extent of trait combinations. This\nsuggests that monocot pollen forms are highly dispersed but within a more\nrestricted region of the morphospace.\n-The ANA grade, Chloranthales, and the other superasterids groups have the\nlowest contributions to the overall disparity, however, to my knowledge those\ngroups contributed relatively high to the overall floral disparity (see Lopez-\nMartinez et al 2024), why?\n>>> For example, the “other superasterids” group shows the highest disparity level\namong eudicots but contributes little to the overall disparity of eudicots. As Foote\n(1993) noted, a subgroup’s disparity and its contribution to total disparity are not\nalways correlated. Groups that lie at the periphery of morphospace and span\ndistinct regions tend to increase overall disparity more. In contrast, the other\nsuperasterids, though internally diverse, occupy a region already densely filled by\nother eudicots, resulting in limited impact on the total morphospace expansion.\nAs for the contrast with López-Martínez et al. (2024), which focused on floral\ndisparity, we agree that this is an interesting result. The difference likely reflects\nthe use of different morphologic systems—pollen versus floral traits—which are\nshaped by different developmental and evolutionary constraints. Disparity patterns\ncan vary depending on the morphological domain under study, and such variation\nis to be expected. We have contrasted our pollen disparity results with the floral\nanalysis of López-Martínez et al. (2024) on lines 298-309.\nRefs cited in this Reply:\nLópez-Martínez A. M., Magallón S., von Balthazar M., Schönenberger J., Sauquet\nH., Chartier M. Angiosperm flowers reached their highest morphological diversity\nearly in their evolutionary history. New Phytol 241, 1348-1360 (2024).\nFoote M. (1993) Contributions of individual taxa to overall morphological disparity.\nPaleobiology 19, 403-419.\n-The comparison between pollen disparity and pollination mode was particularly\ninteresting. The authors obtained the data of pollination mode from Stephens\net al.; however, to my knowledge, the sampling might be substantially different.\nStephens et al. included about 1200 extant taxa, with pollination mode mostly\nscored at species level, which contrasts with the sampling presented in this\nstudy. The authors should clarify these differences and explain how they\naccount for the variation on pollination type within a Genera (e.g., Aquilegia\nshows vertebrate and insect pollination or even exhibit a mixed pollination\nstrategies).\n>>> We clarified this in the Methods that Stephens et al. (2023) compiled\npollination mode data primarily at the species level. In fact, we initially assembled\ngenus-level pollination mode annotations for all sampled taxa using the TRY and\nBIEN databases, supplemented with information from related literatures. Our\npreliminary analyses based on this dataset yielded results that were highly\nconsistent with those derived from Stephens et al. (2023). Given the challenges\nand subjectivity involved in scoring pollination mode and the overall concordance\nof results, we ultimately adopted the dataset from Stephens et al. (2023) for clarity\nand consistency.\nRegarding the concern about within-genus variation in pollination mode, we note\n18\nthat such genera are excluded from the morphospace analysis, as stated in the\nMethods (lines 687-690).\nRefs cited in this Reply:\nStephens R. E., Gallagher R. V., Dun L., Cornwell W., Sauquet H. Insect pollination\nfor most of angiosperm evolutionary history. New Phytol 240, 880-891 (2023).\nMinor comments:\nThe measure of disparity based on the sum of variances using the NMDS and\nPCoA ordination can be problematic when using discrete morphological\ncharacters. I think it is more accurate to measure disparity based on the\ndistance matrix (please see Gerber, 2019. Use and misuse of discrete\ncharacter data for morphospace and disparity analyses. Palaeontology 62: 305-\n319).\n>>> We appreciate the reviewer’s concern regarding the use of ordinated spaces\nfor disparity metrics.\nAmong the four size-related disparity indices we used, only Mean Pairwise\nDistance (MPD) can be directly calculated from the raw distance matrix, and it is\nalso one of the recommended metrics in Gerber (2019).\nTo evaluate the reliability of ordination-based estimates, we compared MPD values\nderived from the raw distance matrix (i.e., without ordination) and from ordinated\ncoordinates (post-ordination). NMDS-derived MPD values were highly correlated\nwith the raw values (Pearson’s r = 0.957, p < 0.001), indicating minimal distortion\nof the relative structure of morphological distances (see Supplementary Fig. 2). In\ncontrast, PCoA-based MPD showed a relatively weaker correlation (r = 0.737, p =\n0.006), reflecting greater information loss during dimensionality reduction.\nThese results support the reliability of our NMDS-based estimates, which we\nprimarily used. Given this strong concordance, we are confident that our approach\ndoes not bias the results or affect the interpretation of disparity patterns.\nWe have further added sentences in the Methods and Result sections (lines 175-\n179, 635-638).\nRefs cited in this Reply:\nGerber S. Use and misuse of discrete character data for morphospace and\ndisparity analyses. Palaeontology 62, 305-319 (2019).\nSincerely Yours,\nDe-Zhu Li PhD FLS OBE FRSE\nProfessor of Botany\n19"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I understand why the authors chose not to reanalyze their data using the Zuntini et al (2024) angiosperm phylogeny. However, it may be a missed opportunity compare trends in an alternative phylogenetic context. Additionally, the taxonomic groups that are referenced should be described in more detail when first introduced, like “other superasterids”, with direct reference to Figure 3. These grouping are not common taxonomic terms and the lack of definition early on makes it difficult to follow the logic of the arguments made. Finally, I am glad to see that the character matrix will be published and missed that it was part of the shared zip file of data. Perhaps also make clear in the spreadsheet/supplementary files that the character codes are found in Supplementary Table 2. Overall, I am satisfied with the authors’ response. Small comments are listed below. Line numbers from “593629_1_other_0_t222p8.pdf” Abstract Line 44: Should read something more like: “Based on inferences from extant taxa/morphologies” Introduction Lines 76-77: “misleads.. our understanding” does not sound correct (phrase used again in Line 372) Results Line 145: There is no phylogeny illustrated in Fig. 2. Should this reference Fig 1 or 3? Line 191: “provides valuable information to help us understand them” is a vague statement Lines 201-213: I do not understand the comparisons among the taxonomic groupings. Fabids are within the eudicots, so is the high disparity in eudicots due to the fabids? What clade is meant by “the other superasterids”? Are these the stem superasterids? This should be clearly defined and perhaps phrased a little more clearly. Line 219: Similarly, define “other monocots” Line 232: Would extinction affect your estimates of rates of morphological change? Line 282: Can you explain what the significant differences are? Line 283: “abiotic and biotic pollination; wind and water insect pollination” Why make this distinction? Methods Line 538: Palynological identifications are typically of genera because there is often limited morphological differences between the pollen of species of the same genera. It would be good if the authors could confirm that that is the case for the genera in this study. Line 611: Delete extraneous “For”", "reviewer_2": "Dear authors, Thank you for addressing my concerns.", "reviewer_3": "Major comment: I appreciate that my comments have been taken into account in this revised version of the manuscript. I believe that the efforts made by the authors to address the suggestions have contributed to improve the quality of this investigation. I appreciate how the authors presented the results and discussion in this new version. However, I would like to see a further discussion on the evolution of the pollen form. For example, which phenotypes are the most common ones? To what extent might these patterns reflect phylogenetic constraints versus evolutionary convergence? Which characters might be responsible for the saturation of certain areas of the morphospace? A particularly relevant study by Luke Mander (see citation below) explores this issue using a “raw” morphospace approach, although it focuses exclusively on pollen from tropical angiosperm species. For example, Mander demonstrates how monocots and eudicots occupy different regions of the morphospace and highlights associations between various discrete characters. He found that pollen grains observed in nature occupy considerable amount of the raw morphospace (which in a certain way study the evolution of forms within those that are possible). In your case, you found a contrast pattern of occupation. Could this be a consequence of not constraining the theoretical morphospace? Are there combinations included in your theoretical morphospace that may in fact be biologically impossible? I believe this work provides an interesting point of comparison for your study and should be considered, especially since it has not yet been cited. Mander Luke. 2016. A combinatorial approach to angiosperm pollen morphology. Proc. R. Soc. B.28320162033. http://doi.org/10.1098/rspb.2016.2033 Minor comments: L109-110: “and the prevalent issue of microfossil identification bias complicates macroevolutionary modeling efforts”. This statement is irrelevant, as comparing disparity through time does not necessarily require taxonomic identification; it only requires knowledge of the fossil age. L110-111. The excellent work by Gravendyck et al. should be cited https://doi.org/10.1073/pnas.2421470122 L149. And what about the ANA grade? L220. Please ensure consistency in group labels throughout the manuscript. For example, in L211 you use “other superasterids” and here you use “other superasterid grade” L233 Could you provide the value of character state changes per my? Just for comparison with the previous sentence. L304. Avoid “basally”-branching L544. Mentioning fossils here is not relevant for your study. L648 The authors state that they were inspired by the simulations of Deline et al. to construct the theoretical morphospace. However, the procedure they describe for generating the theoretical combinations closely resembles the approach presented by López Martínez et al. In fact, the figures included in this manuscript are quite similar to those shown in López Martínez et al. Therefore, I suggest that this paper should at least be cited in the Methods section to recognize this precedent. L659-660: Same approach as Lopez Martinez et al. This paper should be cited. L688. biotic types, and wind, water, insect, and vertebrate types -> biotic types, and wind, water, insect, and vertebrate types? L 690. For me it is not entirely clear how the authors deal with polymorphic data. As far as I know, Stephens et al. used polymorphism to conduct their ASR. They only treated missing data in correlation analyses between high latitude and wind pollination. Figure 4. Avoid using BASAL in the figures."}, "author_response": "RESPONSE TO REVIEWERS´ COMMENTS\nNote: Line numbers refer to the revised manuscript with tracked changes\n(Revised_manuscript_tracked.docx).\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI understand why the authors chose not to reanalyze their data using the Zuntini et\nal (2024) angiosperm phylogeny. However, it may be a missed opportunity\ncompare trends in an alternative phylogenetic context.\nAdditionally, the taxonomic groups that are referenced should be described in more\ndetail when first introduced, like “other superasterids”, with direct reference to\nFigure 3. These grouping are not common taxonomic terms and the lack of\ndefinition early on makes it difficult to follow the logic of the arguments made.\n>>> Thanks for your understanding. We have now added a paragraph in the\nSupplementary Methods explicitly explaining our rationale for the higher-level\ngrouping scheme and providing clear definitions for the informal groups (including\nG1 and G2), with direct references to the relevant figures. We believe this\nclarification resolves the terminology issue raised.\nFinally, I am glad to see that the character matrix will be published and missed that\nit was part of the shared zip file of data. Perhaps also make clear in the\nspreadsheet/supplementary files that the character codes are found in\nSupplementary Table 2.\n>>> Revised.\nOverall, I am satisfied with the authors’ response. Small comments are listed below.\n>>> Thank you for the positive assessment. All comments have been carefully\naddressed.\nLine numbers from “593629_1_other_0_t222p8.pdf”\nAbstract\nLine 44: Should read something more like: “Based on inferences from extant\ntaxa/morphologies”\n>>> Revised to “Based on inferences from extant morphologies”.\nIntroduction\nLines 76-77: “misleads.. our understanding” does not sound correct (phrase used\nagain in Line 372)\n>>> We have revised the wording. Specifically, in the Introduction (line 72) we\nchanged “mislead” to “distort,” and in the Discussion (line 409) we replaced it with\n“obscure.”\nLine 145: There is no phylogeny illustrated in Fig. 2. Should this reference Fig 1 or\n3?\n>>> The correct reference should be to Fig. 1. We have corrected this and re-\nchecked all figure citations to ensure consistency.\nLine 191: “provides valuable information to help us understand them” is a vague\nstatement\n>>> The sentence was revised as \"Although these axes are based on distance\nrather than real biological variables, correlations with original pollen traits allowing\nus to infer the gradients that each axis represents (Extended Data Fig. 1).\"\nLines 201-213: I do not understand the comparisons among the taxonomic\ngroupings. Fabids are within the eudicots, so is the high disparity in eudicots due\nto the fabids? What clade is meant by “the other superasterids”? Are these the\nstem superasterids? This should be clearly defined and perhaps phrased a little\nmore clearly.\n>>> To clarify, we have added a paragraph in the Supplementary Methods\nexplicitly explaining our rationale for the higher-level grouping scheme and\nproviding definitions for the informal groups. We have also revised the phrasing in\nthe main text for clarity, strengthened the figure references, and corrected\nambiguous citations (see lines 196–199).\nLine 219: Similarly, define “other monocots”\n>>> See above.\nLine 232: Would extinction affect your estimates of rates of morphological change?\n>>> The rate tests implemented in Claddis are based on a likelihood-ratio\nframework that uses the branch lengths of the time-calibrated phylogeny and the\nobserved discrete character data of extant taxa, but do not explicitly model\nextinction. Nevertheless, given the breadth of our extant sampling and the fact that\nour analyses emphasize relative rate variation among clades and traits rather than\nabsolute values, we consider it unlikely that unobserved extinct diversity would\nqualitatively alter our main conclusions.\nLine 282: Can you explain what the significant differences are?\n>>> We agree that the wording was imprecise. “significant differences” has been\nrevised to “statistically significant separation” to better reflect the PERMANOVA\nresults. We have also harmonized this phrasing throughout the manuscript.\nLine 283: “abiotic and biotic pollination; wind and water insect pollination” Why\nmake this distinction?\n>>> What we intended was “abiotic and biotic pollination; wind and insect\npollination,” consistent with the categories in Stephens et al. (2023). The revised\n2\ntext now reads: “statistical significant separations were observed among most pairs\nof pollination modes (e.g., abiotic versus biotic, wind versus insect)” (see Extended\nData Figures 9–10 and Supplementary Data 4).\nMethods\nLine 538: Palynological identifications are typically of genera because there is often\nlimited morphological differences between the pollen of species of the same\ngenera. It would be good if the authors could confirm that that is the case for the\ngenera in this study.\n>>> We agree, and this has been one of the main reasons we consistently used\ngenera as our terminal units. Pollen morphology is generally conservative within\ngenera, and our coding strategy reflects this. Indeed, only ~6.2% of our coding\nunits were polymorphic, which we consider to be an acceptable proportion. This\nstatistics has now been added to the Results. This sentence was revised to “This\nlevel of sampling is also well-suited to palynological datasets, owing to the\nrelatively conservative nature of pollen variation within genera”. See lines 138-139,\n536-537.\nLine 611: Delete extraneous “For”\n>>> Revised.\nReviewer #2 (Remarks to the Author):\nDear authors,\nThank you for addressing my concerns.\n>>> We sincerely thank you for your thoughtful comments and constructive\nfeedback, which have greatly helped us to improve the clarity and quality of the\nmanuscript.\nReviewer #3 (Remarks to the Author):\nMajor comment:\nI appreciate that my comments have been taken into account in this revised version\nof the manuscript. I believe that the efforts made by the authors to address the\nsuggestions have contributed to improve the quality of this investigation.\n>>> We sincerely appreciate your positive comments.\nI appreciate how the authors presented the results and discussion in this new\nversion. However, I would like to see a further discussion on the evolution of the\npollen form. For example, which phenotypes are the most common ones? To what\n3\nextent might these patterns reflect phylogenetic constraints versus evolutionary\nconvergence? Which characters might be responsible for the saturation of certain\nareas of the morphospace?\n>>> We agree that interpreting the high-dimensional morphospace constructed\nfrom discrete coding is a major challenge. To aid interpretation beyond overall\npollen disparity, we also analyzed three subsets of traits (shape, aperture, and\nexine). However, with the current distance-matrix-based framework, directly\naddressing the question of “the evolution of the pollen form” would require\nsubstantially expanded analyses that might go beyond the scope and central focus\nof this study, which is on broad-scale patterns of pollen disparity across\nangiosperms.\nThat said, we fully acknowledge the importance of understanding how\nphylogenetic constraints and evolutionary convergence shape morphospace\noccupation. In response to your suggestion, we have incorporated additional\nanalyses of morphological convergence, which yielded intriguing results.\nCombined with phylogenetic signal tests and our theoretical morphospace\nanalyses, these results provide a more refined perspective on how extant\nangiosperm pollen occupies morphospace.\nWe have incorporated these additions into the main text of the Methods,\nResults, and Discussion (see Results, lines 183–190, 225–244; Discussion, lines\n336–359; Methods, lines 629–632, 651–663). We believe that these revisions\nfurther strengthen the manuscript and provide a solid foundation for future\ninvestigations into the evolution of pollen form.\nA particularly relevant study by Luke Mander (see citation below) explores this\nissue using a “raw” morphospace approach, although it focuses exclusively on\npollen from tropical angiosperm species. For example, Mander demonstrates how\nmonocots and eudicots occupy different regions of the morphospace and highlights\nassociations between various discrete characters. He found that pollen grains\nobserved in nature occupy considerable amount of the raw morphospace (which\nin a certain way study the evolution of forms within those that are possible). In your\ncase, you found a contrast pattern of occupation. Could this be a consequence of\nnot constraining the theoretical morphospace? Are there combinations included in\nyour theoretical morphospace that may in fact be biologically impossible? I believe\nthis work provides an interesting point of comparison for your study and should be\nconsidered, especially since it has not yet been cited.\nMander Luke. 2016. A combinatorial approach to angiosperm pollen morphology.\nProc. R. Soc. B.28320162033. http://doi.org/10.1098/rspb.2016.2033\n>>> Thank you for raising this important point. In our analyses, we explicitly\nrestricted inapplicable or biologically impossible trait combinations. However, we\nrealized that in our initial phrasing we mistakenly described our results as\n“occupying an area,” which implied the use of the sum of ranges (SOR), whereas\nour calculations were actually based on the sum of variances (SOV). In our results,\nthe empirical pollen morphospace occupies 89.6% of the theoretical morphospace\n4\nin terms of SOR, but only 49% in terms of SOV. This contrast indicates that while\nthe boundaries of pollen morphospace are extensively explored, the realized\ndistribution is highly concentrated. Notably, in Mander (2016), pollen grains\nobserved in nature were found to occupy a considerable portion of the raw\nmorphospace. Views through the lens of our SOR results, extant angiosperms\nhave explored the boundaries of morphospace to a substantial degree. It should\nbe noted, however, that both SOR and SOV differ methodologically from the raw\nmorphospace approach of Mander (2016), with SOR being comparatively more\nsensitive to outliers.\nFurthermore, in subsequent analyses of phylogenetic signal and\nmorphological convergence, our results reveal a complex interplay between\nphylogenetic constraints and convergence. Interestingly, despite differences in\nmethodological frameworks and coding strategies, our findings are broadly in line\nwith those of Mander (2016), both in terms of morphospace occupation and\nconvergent evolution. While our approach is based on distance matrices and\nordination, Mander’s combinatorial “raw morphospace” framework provides a more\ndirect perspective on the potential versus realized spectrum of pollen forms. We\nnow explicitly cite and discuss Mander (2016). We also note his more recent work\non floral morphology (Mander et al. 2025), which highlights the broader utility of\nthis combinatorial approach.\nIn summary, we have cited Mander’s study and expanded the discussion\naccordingly, and corrected the earlier misstatement regarding SOR versus SOV\n(see lines 183–190, 336–359).\nRefs cited in this Reply:\nMander L. A combinatorial approach to angiosperm pollen morphology. Proc Royal\nSoc B 283, 20162033 (2016).\nMander L., Julier A. C. M., Jaramillo C. The architecture of floral diversity: a\nbipartite network of flower morphology in the Neotropical rainforest of Barro\nColorado Island, Panama. Bot J Linn Soc, boaf032 (2025).\nMinor comments:\nL109-110: “and the prevalent issue of microfossil identification bias complicates\nmacroevolutionary modeling efforts”. This statement is irrelevant, as comparing\ndisparity through time does not necessarily require taxonomic identification; it only\nrequires knowledge of the fossil age.\n>>> The sentence has been revised to:“and taxonomic uncertainty in many\nmicrofossils limits their integration into phylogenetic frameworks, thereby\nconstraining their use in macroevolutionary analyses” (lines 102–104).\nL110-111. The excellent work by Gravendyck et al. should be cited\nhttps://doi.org/10.1073/pnas.2421470122\n>>> Cited\n>>> Recent phylogenetic studies indicate that the phylogenetic relationship of the\nthree orders of ANA grade is generally stable. For this reason, we chose to only\nhighlight the phylogenetic relationships among the five major mesangiosperm\nclades.\nL220. Please ensure consistency in group labels throughout the manuscript. For\nexample, in L211 you use “other superasterids” and here you use “other\nsuperasterid grade”\n>>> All these now consistently use “other\nsuperasterids/eudicots/monocots/superrosids”.\nL233 Could you provide the value of character state changes per my? Just for\ncomparison with the previous sentence.\n>>> Revised as suggested. We have added the corresponding values of character\nstate changes per million years. See lines 246-248.\nL304. Avoid “basally”-branching\n>>> We replaced “basally-branching” with “early-diverging”.\nL544. Mentioning fossils here is not relevant for your study.\n>>> Revised. We have removed the phrase “especially in fossil and herbarium\nmaterial”.\nL648 The authors state that they were inspired by the simulations of Deline et al.\nto construct the theoretical morphospace. However, the procedure they describe\nfor generating the theoretical combinations closely resembles the approach\npresented by López Martínez et al. In fact, the figures included in this manuscript\nare quite similar to those shown in López Martínez et al. Therefore, I suggest that\nthis paper should at least be cited in the Methods section to recognize this\nprecedent.\n>>> We had previously noted the work of López-Martínez et al. (2024) on\ntheoretical morphospace but had not fully understood their methodology. After\ncarefully re-examining their description, we now recognize that our procedure for\ngenerating theoretical combinations is broadly similar to theirs. We have therefore\ncited López-Martínez et al. in the Methods section.\nRefs cited in this Reply:\nLópez-Martínez A. M., Magallón S., von Balthazar M., Schönenberger J., Sauquet\nH., Chartier M. Angiosperm flowers reached their highest morphological diversity\nearly in their evolutionary history. New Phytol 241, 1348-1360 (2024).\nL659-660: Same approach as Lopez Martinez et al. This paper should be cited.\n6\n>>> See above.\nL688. biotic types, and wind, water insect, and vertebrate types -> biotic types, and\nwind, water, insect, and vertebrate types?\n>>> Revised.\nL 690. For me it is not entirely clear how the authors deal with polymorphic data.\nAs far as I know, Stephens et al. used polymorphism to conduct their ASR.\nThey only treated missing data in correlation analyses between high latitude and wind\npollination.\n>>> In our correlation analyses we did not perform ancestral state reconstructions;\nall data were restricted to extant taxa. Genera with polymorphic states were\nexcluded both from the Pearson correlation tests and from the comparisons of\nmorphospace occupation across different pollination modes (Extended Data Figs.\n9–10). We have clarified this point in the Methods section and also specified the\nexact number of genera included in the pollination-related analyses (see lines 686–\n698).\nRefs cited in this Reply:\nStephens R. E., Gallagher R. V., Dun L., Cornwell W., Sauquet H. Insect pollination\nfor most of angiosperm evolutionary history. New Phytol 240, 880-891 (2023).\nFigure 4. Avoid using BASAL in the figures.\n>>> Revised.\nSincerely Yours,\nDe-Zhu Li PhD FLS OBE FRSE\nProfessor of Botany\n7"}, {"round": 3, "reviewer_comments": {"reviewer_3": "Dear authors, I would like to thank the authors for taking into account my comments. I consider this revised version considerably improved, especially concerning the description of the theoretical morphospace and the comparison of disparity through time to what has been observed in the fossil record. The incorporation of those new analysis of morphological rates and convergence makes the general conclusion of this manuscript strongly substantiated. Thanks!"}, "author_response": ""}]

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