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Research article Open Access Have giant lobelias evolved several times independently? Life form shifts and historical biogeography of the cosmopolitan and highly diverse subfamily Lobelioideae (Campanulaceae) Alexandre Antonelli1,2

Address: 1Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, 40530, Göteborg, Sweden and 2Present address : University of Zurich, Institute of Systematic Botany, Zollikerstrasse 107, CH 8008, Zürich, Switzerland Email: Alexandre Antonelli - [email protected]

Published: 26 November 2009 Received: 29 October 2009 Accepted: 26 November 2009 BMC Biology 2009, 7:82 doi:10.1186/1741-7007-7-82 This article is available from: http://www.biomedcentral.com/1741-7007/7/82 © 2009 Antonelli; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: The tendency of animals and plants to independently develop similar features under similar evolutionary pressures - convergence - is a widespread phenomenon in nature. In plants, convergence has been suggested to explain the striking similarity in life form between the giant lobelioids (Campanulaceae, the bellflower family) of Africa and the Hawaiian Islands. Under this assumption these plants would have developed the giant habit from herbaceous ancestors independently, in much the same way as has been suggested for the giant senecios of Africa and the silversword alliance of Hawaii. Results: Phylogenetic analyses based on plastid (rbcL, trnL-F) and nuclear (internal transcribed spacer [ITS]) DNA sequences for 101 species in subfamily Lobelioideae demonstrate that the large lobelioids from eastern Africa the Hawaiian Islands, and also South America, French Polynesia and southeast Asia, form a strongly supported monophyletic group. Ancestral state reconstructions of life form and distribution, taking into account phylogenetic uncertainty, indicate their descent from a woody ancestor that was probably confined to Africa. Molecular dating analyses using Penalized Likelihood and Bayesian relaxed clock approaches, and combining multiple calibration points, estimate their first diversification at ~25-33 million years ago (Ma), shortly followed by several long- distance dispersal events that resulted in the current pantropical distribution. Conclusion: These results confidently show that lobelioid species, commonly called 'giant', are very closely related and have not developed their giant form from herbaceous ancestors independently. This study, which includes the hitherto largest taxon sampling for subfamily Lobelioideae, highlights the need for a broad phylogenetic framework for testing assumptions about morphological development in general, and convergent evolution in particular.

Background were certainly like the fish, but it also had mammary When the great 18th-century naturalist Carl Linnaeus took glands and lungs and should, thus, be classified as a mam- a closer look at a the whale during the preparation of his mal. Ever since Linnaeus, what we today refer to as conver- natural classification system, he realized for the first time gent evolution (see, for example, [2,3]) has been that it was not a fish but a mammal [1]. Its form and fins advocated as a common and widespread phenomenon in

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nature. From single features (such as the wings of birds ently from herbaceous ancestors, it seems vital to include and insects or the cones of cycads and conifers) to whole a considerably larger representation of herbaceous taxa organisms (shrimps and krill, cacti and euphorbias), and to root the Lobelioideae tree with representative taxa superficial similarity has been repeatedly demonstrated to from outside the subfamily. evolve independently in distantly related evolutionary lineages. Here I build on previous studies to reconstruct the life form and biogeographic evolution of Lobelioideae. I In plants, the giant lobelias from the Hawaiian Islands focus on the following questions: (i) have giant lobelioids and tropical Africa have been cited as remarkable exam- evolved from herbaceous ancestors several times inde- ples of morphological convergence in the family Campan- pendently?; (ii) when and where did the giant habit ulaceae (the bellflower family). According to some earlier evolve?; and (iii) is the life form and geographic distribu- authors [4-6], convergence from herbaceous plants into tion phylogenetically conservative? To address these ques- tall treelets would have occurred independently in differ- tions, I generated fast-evolving nuclear sequence data for ent mountain systems in response to similar tropical several giant species and, in combination with all availa- alpine climates consisting of nightly frosts and rapid tem- ble DNA sequences for selected markers, I performed phy- perature fluctuations. The idea that many morphological logenetic, biogeographic, character evolution and features in giant lobelias represent adaptations to proxi- molecular dating analyses. The results reported here dem- mal environmental factors [7] is based on the observation onstrate that the giant lobelioids have a very different evo- that the leaf rosettes of many giant species provide insula- lutionary history than the giant Asteraceae. tion for the central axis, protecting the shoot apex and the young leaves and inflorescences from extreme tempera- Results and discussion tures. According to Hedberg [4], a similar evolutionary Phylogeny pattern can be observed between the giant senecios of The maximum likelihood trees based on all Campanu- Africa and the silversword alliance of the Hawaiian laceae sequences available at GenBank and comple- Islands (both in Asteraceae), which Hedberg cited as an mented by novel sequences generated in this study for example of convergent evolution alongside the giant trnL-F, ITS and rbcL are shown in Figure 2a, c. Lobelioi- lobelias (Figure 1). deae is retrieved as monophyletic in all three analyses of each marker separately, providing an indication of the Since these pioneering works, many studies have choice of taxa for the subsequent analyses. The results addressed specific aspects of giant lobelioids, not only from the Bayesian and bootstrap analyses on the Lobelio- providing novel insights into the conspicuous habit of the ideae data set are shown together in Figure 3. Generally, giant forms [6] but also their detailed morphology [8-10], strongly supported relationships were in agreement with, chromosome numbers [11,12], conservation status [13] or at least did not contradict, previous phylogenetic anal- and physiology [14-17]. Based on a phylogenetic analysis yses [7,8,10,18-21]. The few differences obtained here of 17 species in subfamily Lobelioideae (including 13 (especially in clade N4, Figure 3) are discussed below. giant forms and four herbaceous taxa), Knox et al. [18] proposed that the giant lobelioids consisted of a Chilean Molecular dating hexaploid group and a pantropical tetraploid group, all The results from the molecular dating analyses are sum- derived from herbaceous ancestors. Later, using the Chil- marized in Table 1. Generally, node age estimations ean giant species as outgroup and analysing the relation- exhibited relatively large confidence intervals, both under ships and biogeographic history among 21 African and the Penalized Likelihood (Figure 4) and the Bayesian one Brazilian giant lobelia, Knox and Palmer [7] sug- relaxed clock (Figure 5) methods. The tree with highest gested that the eastern African giant lobelias arrived in sum of clade credibilities generated under the Bayesian Africa from the Asian/Pacific region. More recently, evolutionary analysis by sampling trees (BEAST) analysis Givnish et al. [10] used a larger data set (including 38 (Figure 5; effective sample size = 322) did not exhibit any lobelioid species, of which two are herbaceous) to dem- strongly supported phylogenetic conflict with the consen- onstrate that all Hawaiian lobelioids constitute a mono- sus cladogram of the MrBayes analysis (Figure 3). phyletic clade, thus corroborating previous results by Although there was great variation between the age esti- Givnish and co-workers [8,19] mates obtained in Penalized Likelihood as compared to BEAST, in all key nodes listed in Table 1 there was consid- Despite these major advances, we still need to verify erable overlap of the 95% confidence intervals and high- whether the biogeographic and phylogenetic patterns est probability densities. The mean covariance between obtained so far for Lobelioideae will stand the inclusion parental and child lineages in the BEAST analysis was of increased taxon sampling. In order to confidently test 0.157 (Figure 6a). Since a covariance close to zero indi- whether the giant forms worldwide evolved independ- cates no significant autocorrelation of rates (the funda-

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SameFigure life 1 form, same history? Same life form, same history? Giant lobelioids (Campanulaceae: Lobelioideae) from the Hawaiian Islands (a) have been sug- gested to have converged into the giant life form independently from the giant lobelias of Africa (b), in much the same way as the silversword alliance of Hawaii (c) and the giant senecios of Africa (d) in family Asteraceae. [Credits: a, Lobelia gloria-montis by Frederick R. Warshauer; b, Lobelia rhynchopetalum by Christian Puff; c, Argyroxiphium sandwicense by Gerald D. Carr; d, Den- drosenecio keniodendron, from http://www.wikipedia.org].

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mental assumption made by Penalized Likelihood), this

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na DQ285167 Lobelia vivaldii 8 br 51 a an a D 2 6 rmon a bu D t n g a A 6 8 indicates that the BEAST results reported here are proba- 8 7 r ti C y s D 5 51 C s Tre 8 1 u l D Y l ti A 6 2 r e ar a hi n n s Y 7 C o a m s 1 ya p i D 6 9 n tr c s 5 u DQ3 a e a 5 6 1 u u e 5 DQ 9 r ra mat 9 a ka m Y 8 Ce o pa A 6 c u 53 9 L il ti i s 5 1 e n a mp a e 1 y n a e nd th s A 5 5 6 5 l f D 1 b AY Y 2 B e c ne o i a 5 o c a r id m 5 0 L o an k i A Y 6 5 nt t y 5 e n a AY5 6 s w oresc ke n Y 5 e Q lep r r a A 3 h c s 2 o b a s Q 5 2 4 C e t p n u io T atol 1 L a ti v te s a A i l ea ol a a Q3 Ce p ho l s D 2 be e e a ke a A 2 1 5 i s r o i a i D 51 C i m y e DQ356 L L fl t s 5621 7 ho u Q D 8 e o li k l D Q3 2 l ac b a a tos f a 8 6 e a p b o ob a n l DQ 3 51 m b l u a or a 6 21 1 0 S p ogo y a A 35619 Q o e n aur D 5 Sip i rme c DQ b i c a Q35619 er e a hihew e DQ3 6 2 m p m p 3 at be e l h g o n a Q 3 5 S u r a d 62 lia e i t D Q2 62 18 3 o a t foli 5 li a h y u eg ns a 3 5 B u ho m ia lo e i D olob l r i D 5 62 2 2 tr c a ti i 3 6 l i a p s i D 3 a g h ia a y yp a e Q B ip n o r s h Q 18 5 2 Lo ma ol t i DQ 3 5 6 2 22 4 c ili y u c a i h u 6 62 n if D 3 5 6 2 6 3 S e o e ic a 2 0 m c o e ea c g a 6 be elia i u o a DQ 3 6 1 C ip h b gia n n D 8 Lob 1 L cr i c l u Q 1 4 a n re ia 00 ac h c e l DQ 35 5 5 p ll n a a ri l Q 5 L c o c ia Q 2 2 6 S i a ld t D li os a uc D 3 e n g o D 1 ob ob o i a 8 6 2 le e n Q 3 a ros u d D 2 6 S a a lif Q D 5 el L ka ide Q 2 5 6 2 i h o t s D 3 5 8 e or t e e a DQ 1 1 r u Q 6 i o e a n Q 3 5 6 Ro m n ere e 2 Q 5 Lo a les l b ua tac c s D 2 2 e lenber o p m DQ 8 2 1 ia li g s 3 5 6 9 Wah um id 5 2 6 8 e a f hys s D 2 e ne e 8 8 1 st a ie is DQ Q 3 5 6 8 3 F n m la pri a le lo D 1 5 7 b li n hys u 28 51 9 e a i D Q 3 5 7 9 u e r u x Q 5 16 2 L c r stulosens n io n r r y 5 li he ic e si D Q 3 7 80 asio s ne u e e l a 2 5 Ap 5 Lo o a 88 8 J a o a ula a a p s D 1 7 0 b c kl xa s D 0 7 p n c ca 8 6 L naultiana 8 1 J si s i lo es Q28 5 L be e a is DQ a ia b d 1 9 e ob l o n ltat 8 4 a m p m a u ides i t o ia l a EF 8792 Wah 7 J a u u ic 5 Apeta a b li u di m ch li m p t 5 6 h e e a gib s m a EF08F08 08 87 1 2 C a u u ino tho a D 1 lia l a 6 8 ss e li la r r l Q 7 S ia ia a n e E 08 2 h f u D c t a 5 7 4 C c e u pa 2 Q 0 l g ti EF 8 9 Mu h n la a bly more realistic than the ages obtained in Penalized 8 le h on b m r 8 7 a a ca 2 Sc r i o ibberoabe is EF 8 r ac u a o ii D 5 8 o a gi n a 0 8 69 T r p n l r ia D D 1 l t m r e DQ3 F 85 a e Q Q 5 5 er h stigmataine oa 7 1 T m nu la prenant en fol DQ 2 Q2 0 1 e E F0 88 8 p i lia 2 4 o c a 0 79 0 mp a a tev ic D 8 8 8 L 8 th r n E F 8 7 Ca a p l s s fo 2 5 51 51 o a g s 0 8 2 u r lia 8 Q 1 b Lob eca j ar is E F 8 61 C m panu n e ici 5 3 6 7 e a 5 a a s ifo m D 1 5 1 6 6 y e E F0 75 4 C p ula p r u Q28514 6 Lobe3 lia e f o ta 3 8 2 am a ic n 6 1 L L l o ru E Q 8 7 2 C m an ul pe rs 8 o o k ia rste e 0 8 a p n a D D 8 b b a m D 8 a la pe rica Q e D lia eli e u g 0 C p rid e 4 li li a lo r EF F 8870 748 1 nu b m 35 7 s e a a e ri i 8 5 Cam m ula da a D 6 se li n v ns a E F0 7 a pa n hy D 1 D s ico p i 8 m a m a Q2 8 eli a s e ll i E 8 C a ia ybri u t Q28 9 r ea t tio o s F08 9 h r a 8 B s hy ia s E 4 C mp us a st nn a 5 se u n lata a F0 88770 3 a o i la i at D 51 1 ri a tid n if E 0 31 C s u p n a m D Q3 40 gh su o d o F 1 74 3 eg ou n la in s iu Q 39 s ul li E 2 L g a u o l 3 Brigham p at a 57 p r a p ifo 5 5 B b e EF 6 34 a om ri 6 61 ri ia corda rm a F088 2 m m ul la D 19 8 gh E 42 3 Le o ar s c u D D Q 4 4 amiaam in a J 56 Ca tr m xi b m Q Q 35 L s F 3 78 6 e e lo tu 3 3 Lo o ia igni ta Q 88 7 P tro pl g a ra 56 D 56 6 b b ro D 0 7 5 e o a pic lo m 1 Q 1 233 L e eli ins ck s F 88 8 P s m s u 68 3 95 lia a E 0 5 6 hy u a nif ri in 56 L a ig ii 6 te u e c s Likelihood [22]. For comparison, both results are DQ G g 2 P m ly e ra 18 o o r n ni EF 4 78 y u la a id D 3 2 be b an c s J 88 86 h te u lo Q 56 m eli i ep F 5 P y an pichl oc A 356 m Lo lia a tic s F0 6 2 h a ch nu s Y 18 a b d eri o E 2 58 P mp m tri pa n 56 18 0 to e e la J4 6 7 a eu a m e D 8 1 L the lia p n F 2 78 C m a sc s Q 745 Lob o a re us 4 4 u c e e 35 be ca q ss FJ 88 7 Asyn e a an id E 6 C e li b u a 0 5 n m c io s E F0 167 o lia a e a 26 69 sy eu a bel e F 8 le tom rg tic EF 4 A n m lo id 08 877 C ns co ia a J 65 6 sy u lio ium 8 an o ro en n F 42 56 A e ma be ol 78 7 C a a n t a J 6 0 yn u lo if m A 8 P a ri ph opi os F 2 7 As ne a on iu J4 n na y f a 4 65 7 m im ifol 3 laty ar c sa ol FJ 2 6 sy u l n lora 0 c in a lo ia J4 5 A e a o tif 967 od a c na id F 26 95 yn um lim o on a ri e 4 6 As e a m ta He g na en s FJ 88 8 n re ia li ran ri si 0 56 Asy um ora tig ria anthu en s EF 6 1 yne h s la dif si 42 7 op a fa ba s lo s J 65 4 As en ul rme an rus F 42 9 an cym o n 86 Ad p la osif na u FJ 08 93 am u m a us F 86 C pan co na agi E 8 1 m chl F0 2 a uma eati ns E 887 5 C ne a r rte ta reported throughout the paper. 1 y ul o lla F0 87 As p tre E 08 mpan ula es F 572 a an en ica E C p la f an ta J426 589 m nu arg lla F 7 Ca pa la g stre 58 am nu ne sis FJ42626 9 C pa fe en J4 7 am nula bar ca F C pa de leni J4265 145 m la al F 13 Ca anu eph 2 84 mp c ica EF 65 Ca nula gan J42 5 pa gar ca F 657 am la ani ana E 42 C panu rg sky F21 FJ 576 am la ga har a 317 426 C nu osc an EF213 4 Ed FJ 725 pa a p rsky rai 088 am nul ha E 175 an EF 1 C pa posc F21 Edra thus 2658 am ula nica 317 ian au J4 88 C pan sita EF2 8 Ed thu stra F 65 la lu 131 raian s au lis J42 7 Cam u rsa EF 76 Ed thu stralis F 874 mpan spa lus 21 ra s a F08 Ca ula ncu 3167 iant ustra E 3 pan pu EF2 Ed hus lis 8873 Cam ula ra 131 raia austr EF0 59 pan ula 65 E nthu alis 131 am pat EF21 dra s au EF2 58 C nula na 317 ianth stra 887 mpa bieti E 9 Edr us a lis F0 Ca la a ii F213 aian ustr E 148 panu djan 181 Ed thus alis 213 am hara EF2 rai aust EF 97 C nula 13168 anthu ralis 0886 mpa pica E Ed s aus EF Ca a olym ns F213 raianth tralis 8726 panul umbe 186 us EF08 Cam dec a EF Edraia austra 8741 anula inian 21317 nthus lis EF08 amp aldste 0 Edr aust 716 C ula w na EF21317 aianth ralis F088 mpan asinia 2 Ed us aus E 63 Ca omm EF raian tralis 2131 nula t 213183 thus aus EF Campa sicolor Edraian tralis 426590 ula ver s EF21316 thus aus FJ Campan amidali 9 Edrai tralis 26591 ula pyr EF2 anthus FJ4 ampan lla 13171 E austral 8754 C isophy draianthu is EF08 panula EF2131 s austr Cam lis 85 Edraia alis FJ426583 a fragi Life form shifts nthus anul EF2131 australi 580 Camp s 82 Edraian s FJ426 ula elatine E thus austra 7 Campan F213184 Ed lis FJ42657 latinoides raianthus mpanula e EF21 australis 26578 Ca kinsiana 3177 Edraian FJ4 anula haw thus australi 3146 Camp EF213180 E s EF21 ochlearifolia draianthus austr Campanula c EF alis EF088710 213173 Edraianthus a anula parryi ustralis EF213147 Camp EF213166 Edraianthus divaricata australis EF088718 Campanula EF213277 rotundifolia Edraianthus horvatii EF088759 Campanula i EF213278 Edraianthus horvatii EF088762 Campanula scheuchzer EF213276 Edraianthus horvatii EF213164 Campanula witasekiana EF213275 Edraianthus horvatii EF213153 Campanula rotundifolia EF213279 Edraianthus horvatii EF213154 Campanula sp aianthus graminifolius EF088740 Camp The character state reconstruction of life form is shown in EF213205 Edr anula moravica s graminifolius EF0 EF213203 Edraianthu 88765 Campanula scutellata hus graminifolius EF08876 213202 Edraiant 6 Campanula semise EF raminifolius E cta draianthus g F088737 Campa EF213199 E us nula macrostyla us graminifoli EF088772 Cam 38 Edraianth panula strigosa EF2132 raminifolius EF0887 raianthus g 94 Campanu 213237 Ed inifolius EF0 la propinqua EF thus gram 88749 Cam 9 Edraian ifolius E panula pr EF21321 s gramin F088778 opinqua aianthu Diosphaer 245 Edr minifolius EF0887 a rumelia EF213 thus gra 44 Campa na Edraian nifolius EF088 nula pinat F213246 s grami 714 Ca zii E ianthu culus EF0 mpanula 248 Edra nthus si 88719 C creutzbu EF213 draia ifolius EF0 ampan rgii 13317 E min 88720 ula drabi EF2 thus gra lius EF0 Campanu folia draian minifo 8869 la e 3201 E gra us EF 6 Azo rinus Figure 7 (see Table 2 for the definitions). The reconstruc- thus li 08 rin EF21 draian minifo 8746 a vidal 79 E us gra ius EF Cam ii EF0887 aianth inifol 0887 panula 0 Edr gram lius E 08 Ca polycl 21326 anthus o F088 mpan ada EF drai graminif us EF 705 C ula di 259 E thus inifoli 088 amp morph F213 raian ram 717 anula antha E 7 Ed us g olius EF0 Cam balfou 1322 ianth minif 8873 panul rii EF2 Edra gra lius EF 9 Ca a d 200 thus inifo 0887 mpan ichot 213 raian gram lius EF 22 C ula mo oma EF 1 Ed hus nifo 088763 amp llis 325 iant rami lius E anu F21 ra us g nifo F08 Cam la fili E 11 Ed nth i s E 876 pan caul 2132 raia s gram oliu F088 9 Ca ula is EF 2 Ed thu nif 70 mp scle 321 aian rami lius DQ3 0 C anu rotri F21 Edr s g inifo 56 amp la spica cha E 41 nthu am ius EF 171 anula 32 raia s gr nifol 088 Ca ta EF21 Ed thu i lius EF 774 mpa alliar 53 ian gram ifo 21 Ca nul iif 2132 ra hus min ius E 334 mp a tr olia EF 9 Ed ant ra ifol F08 2 S an ache 324 ai g in E 87 ym ula t liu F21 Edr thus ram lius F0 04 ph rac m tion of life form on the Bayesian cladogram required 68 E 4 an g fo 8 C y he 321 rai us ini EF 87 amp and lium 21 Ed nth am olius 0 23 a ra EF 33 ia gr inif s E 887 Cam nu zan 132 dra hus m liu F0 84 pa la a gez F2 9 E nt gra ifo s 88 M nu rme ura E 22 raia us min liu EF 73 ich la fo na 13 d th ra ifo 08 5 C au li 2 63 E ian s g in EF 87 am xia osa EF 32 dra thu ram olius 08 29 p tch 21 E an g inif s E 87 Cam anu ihat EF 258 rai us m oliu F0 12 la ch 13 Ed nth ra if EF 88 Ca pa ma ew F2 4 aia s g min lius 088 71 m nul cr ii E 26 dr thu ra ifo s E 3 C p a i oc 13 E an g liu F 77 a an nv hla 2 62 rai us amin ifo s 213143 1 C mp ula olu mys EF 32 d th r in EF a an co cra 1 1 E ian us g am 088 m ula nf ta EF2 26 dra h gr inifoliu s EF 7 C pa c ert 13 E iant s oliu 08 11 am nu ori a F2 54 ra thu ram if E 8 C p la ace E 32 d n s g lius F2 75 a an str a 1 E aia hu min ifo s E 13 3 mpa ula icta EF2 65 dr nt n liu F0 14 Ca col 32 E ia ami ifo E 88 4 m nul l 21 71 a thus gragr in lius F0 7 Cam pan a ina EF 32 Edr an s m ifo s E 8 09 u co 21 3 ai gra in F 87 Ca p la llin F 27 dr nthu m oliu s E 21 9 an pun a E E ia us ra nif u F 31 6 mp ul 13 6 h g oli E 2 62 Cam a a cta steps and the character is phylogenetically conservative (P F2 dra nt s mi if s 1 n d E 25 E a u ra in liu F 31 C p ul as ta ai th g m o s E 21 5 a a a ya F213 274 s a nif liu F 3 0 m nu ch n E 3 Edr aian hu gr i o EF 21 15 C pa la a th 21 70 dr s am if s 31 1 am n t mi a F E iant u in liu 2 C p ul ym sson E 132 7 ra th gr m ifo EF 13 61 am a a p 2 5 d an us ra n us 2 2 C nu ty h i EF 32 E ai th i oli s EF 13 8 a pa la m aea s 1 2 r s g am if 2 2 7 m nu r ph F2 27 Ed an hu r veu s E 1 8 E pa ad a E 3 9 rai t g min i iu F 3 4 dr la ic ea 21 6 d an us ra n ol s E 21 28 E a nu ra o F 2 E i th g us if liu F 32 6 d ia la d sa E 13 dra n s h in o E 21 E ra nt ty ic 204 ia hu nt m f F2 3 85 d ia hu m os F2 6 E a t ia a ini lius E 1 ra n s a E 13 0 dr an gr m fo us F 13 57 C E i th p ph F2 2 E i dra s i E 0 1 dr an us ar a E 13 7 ra E u gra in us F2 8 58 a th p n ea 2 0 d 1 h m li E 87 a ian u a a 32 E nt s ra o F 1 6 C m s rn ss EF 1 36 28 ia hu g if E 21 3 7 am p th par as ic 2 2 3 a in folius F 28 C a u u F 3 21 dr ant us m i EF0 2 3 2 Edr a pan nu s n sic s E 1 E i s graminifolia in olius s 1 28 m l pa a u 2 EF ra nth u r m if iu E 3 a r s s 4 h g a n l 8 1 3 p u s n si EF 4 ia t s r i ifo ius F 87 5 E a a la ib a cu 32 Ed ra u g m n l s E 0 2 C d i nu s ir ss s 1 30 an th s a i iu F 8 5 r ant l ib ic ic 2 Ed rai n r m l E 0 879 7 a a ir a u F 32 3 d ia thu a o F 8 a ia h s ic s E 1 2 E a s g r minifoif E 2 87 5 Ca m n u ib a 2 2 r an u g a in olius us F 1 p th s ir < 0.001; Figure 8a, b). The ancestral life form of the most d i h i E 6 m o F 13 a s r m nif l s 2 3 1 C a us ic E 2 225 E r u g a fo u F 1 1 a p n w a F 5 iant th r mi li s E 0 3 6 C m a ula o er E 13 3 Ed ra n g ini fo E F0 8 1 0 a p nu w in 2 0 d ia thus s ra i s 8 5 C m a r er i F2 3 n u g m in EF0 F 8 728 5 am n la r a an E 1 E ra h s s 0 8 C pa u p in 2 d a t ra m inifoliuoliu 8 7 l a u i us 1325 32 E n g a if liu E 8 03 C a p n a p n a EF thu s r m n o us EF F0 88 7 C m a u s u c n 5 g a i if s 0 a p n la a n u us 132 1 raia s r iu E 8 7 1 m an u x c lo EF2 2 2 6 Edraid u ifoli 0 8 6 C a p la s if u id 3 6 raian nthu h s g ram min ol E F0 8 7 0 m u a ra lo es EF 1 2 E t g f s 8 4 a a la t xif i 2 3 aia n ra iu EF F 8 7 C p n r g d F 3 4 Ed a thu s ini foliusl s 0 8 5 m an u id ra a e E 1 2 i u g amin o E 88 56 a p l s e s 3 a n m ini if s 0 7 C m a a n ga 17 r ia th a E F08 8 6 C a a ul in xi 1 2 d n us gr m in foliu liu s 7 4 m p n a f ta EF2 2 ra h s gr a u E F 87 2 a u cur r t F E ia t r m ni o s 0 C a p a a a 13 7 a n hu s i if li E F 8 7 m n la g E t a s 8 7 3 a an u rg va a F2 6 Ed dr ia r m in ifo eu EF F 0 8 1 Ca p la a 132682 Edr n hu s g a v 8 34 m an ul n a E 2 3 E ra ia u g r m in i liu EF 0 8 7 Ca e F nt s n o ius s 8 5 m pa a s d a 1 221 6 a th g a m f E 2 76 C u a r E 2 1 r ia u r s s 1 8 5 mp pa n p e F Ed d n s g a ni E F2 0 69 a la v ws 2 4 ra th u r u iu 8 3 8 Ca u in a E 213 3 E ia n s oliu l s EF F 1 m nu l r l 3 d a h g th mi f u 873 8 Cam a a ad n an i 1 2 1 ia t u E 2 1 4 m p a i EF 2 3 E n h s a ini ifo s 3 n la s t u r E F0 2 13 1 Ca a ul u t ic F 1 0 ra ia aminifol lius u s 3 p n c if Edr h g r m in ifoli i EF F 13 8 Ca a h o la i a E 323 2 d a t aian fo l u s 3 3 m a u d F2 1 2 8 ian r a m in i o i E 2 8 9 p n o p a E n g r f l u 3 4 C an la la fm a E 2 3 dr a a in i o i s E F 2 1 8 P mp p u 1 8 r ia g r m f l 4 1 a na r recent common ancestor (MRCA) of Lobelioideae (N1 in F E Ed hus n i u e E 3 7 a l ly i 2 2 d t us g a i o li F 08 1 0 mp ul a an a E ra n f a e E 1 07 C Petko e n F 2 n h us i o r F 0 3 Petko an q r t E d t s m EF 4 t ul a a a 213213 39 80 ia n if a F 0 8 8 1 k u n E th u a mi i t EF 2 ov a t 1 2 E 2 s gr gram r n 08 8 8 7 5 ul a s e a ii 3 ian h i ara s D n p EF 2 2 t m 0 8 7 7 6 af r 1 242 dra a ra t s EF C a i u a ec cet F 5 13 r n a m tarae iu Q 08 8 73 5 5 via a 3 2 thu us s g g r us s l s E 8 C a mpa la a fin E 1 2 E aian a a h tarae iu s E 8 7 0 F2 r i n u g o l F 3 C m vi or l i 3 r t f iu s EF 0 a p os o 2 Ed a i l u F 8 7 0 0 o m is E h us n s E 2 5 C m a r F 1 EF 24 r ia t s o li u 8 7 a p p e g u ifo i s E 2 61 3 6 e a um d a anth u f o l u F 1 8 C a m n o r ha s E i n s ia u i s E 2 7 6 pa a p 47 r hus n f li u F 1 u d F2 E a h t i i s E 33 7 C a m n r tr d a i u a n u l E F 2 1 3 p h r t e o s 33 69 l p i is E 132 r anthus t ifo liu u E F 2 32 C m u a n u a anth n h aianthu e n f s 1 3 a a nu a 32 6 d i t i u EF F 2 C p ha i m r r u fo li E 1 02 l ai e o i s F 2 3 amp m n a ni d 1 2 a s t l E 1 2 p a b F2 3 E E d i n r t u f o us 2 01 3240 Ed ra Ed i liu E 1 3 C C a l e 2 aian u s i u s EF F 2 3 n u ni a tenu n o 33 9 pa a a b et d E 1 1 E i Ed en u if f us EF F 2 13 3 m a F2 3 ra h s li E F 3 0 u l 9 nui i u EF 2 1 9 Ed am n a e 2 e fo ol s e de a Ed dr t t u EF2 2 13 0 a se u 1 22 d n i s E 3 0 Ed E 0 1 c us s E F 2 3 ul l a u E 13 p a nu t F2 3 thu t if s E 1 a li n s n cus E 2 30 m us s EF 1 4 Edr p 2 E u i E u EF213196 Edraianthus glisicii

2 e EF213190 Edraianthus glisicii ifo s E 2 1 E 3 bic i F 1 dr 18 te u s i 1 a a i 1 fo 9 Ed E thu s t n ic i F 2 2 r n u cu u i r n F E a k E u i 1 p i 2 tar F 3 i 3 l u 2 ia n F2 p 2 3 bi u 1 0 a a F pa 3 s F r if th te icu F n l 21 u 2 3 89 E u 0 e ni 28 n u n b c 2 1 8 a ic n a a li 1 n 3 c 2 13 5 E s t er n 3 8 d a t i i 21 EF 3 1 i 1 E i g o r i 3 th i u i 2 u n ic c erbi b 2 31 la 2 1 a 2 a a h te ic 3 a 0 ra 1 E d a nu s b i m 5 i 13 3 E r i 1 2 a t u r b ei 1 9 E a l l te enu e s nu b 13 1 3 n e e 31 a l h t d s 131 32 s s r b e r ia 1 i EF t 3 ai 1 8 nthu 2 1 1 i r ia u r t 3 i a 5 t ser t o 2 t 19 1 E d r m s r 4 e l a 2 t s at 3 3 a n m e i 13320 s 3 r e s 5 a 3 ra th 3 19 s 3 d c s ia 3 9 la EF2 2 a h s e s 30 s an F t 30 6 r a an u s e E n t g s ll F 2 t s s 3 d i t l EF Ed t 0 3 r 3 s t i 1 d i hu a k i u 3 E r a 1 a a t 3 s s m 1 4 dr h a c f an t t i E h s ma 3 E ia t u F21 F 213321 n 0 dr r a d u o hu l 6 Ed 2 E a s 07 Ed l 92 l t et ia s h i E 1 1 n cro 1 9 e u s 9 d a u ian t e 9 t a r nt e s b f 7 Edr ai th hu s 3 ia i sc l E F a 8 E d n E a ai h a o F n u s E u f i 2 u t i t 1 s h 5 r n thu u 7 ra i t d a a w Ed ai n ca a w o 2 0 n u h 332 t dal w E a i w ra t us s ti i l d E E E r h u d s F E n a h n f ra i

Edr h E dr an h h t 3 4 Ed ia t E s s li a 3 3 a Edr ra u t er o E r h E n fo ia n E a s E ant t d r ia h i h s th nthus serbi t s s d p E ianthu s u a 2 8 Ed a an s d i s er ta e 1 3 3 ia t d r a s s li d a a n dr a t n u i u d r ai u d 6 r a d l i Ed n u u ra i a h s p u a n t s e n 3 3 dr u r a ta 1 r a r a i a u i a n h chy 9 E ra h s p a ian r ai a d h r 2 r e i y a s r ai a h h n u r 32 ia h ia t h dr h t ai an ai F2 E t a i n s 5 aia i a s t a Figure 3) is ambiguous, due to an early split between a t i t h u e py y t 3 a ian t an n r l is a s 13 3 a i a t h u s a F2 n an erp lif 2 E r n i a t py a i E i n E n h r l a u s r Ed n r n a s a a t 3 3 Ed a a h erp l 2 2 r n t us s p a F21 Ed a Edrai r h d if E 3 2 d a n t l ol ant a u d n s e 332 a a nt h d i t l 1 3 1 Edr n n i i n h u p d i t 3 4 i y o Edr t l i E 3 6 d hu u i 2 t h s p f E t rp l 2 ai h us n iu 3 9 a E t yl t s a t E a h u s u l i o 3 a h l a EF E h 1 9 h s fol h l r 3 h s u yl ius 21 E r us 31 5 r a F 3 2 r us s e mi i 1 33 u s l s EF213 2 us yll l fo 2 93 dr us s s 2 8 u e i u mi 3 d 4 u i d d g r l us 1 d 3 7 erp rp fo E 2 9 s g i e i i F 3 9 s r 1 s s 33 9 s fo l u 2 29 E g l c EF Edr E 1 9 l i E E s p i 2 98 l i gl s r i f l 1 e 1 s l u 2 i y E s s o u s 1 l i o i F 2 13290 2 s p g e g s y li 3 2 e is o u 7 EF 7 e 2 6 r l s F2 3 5 3 e i yl 2 8 s li 2 13 3 e i c yl ll l u F l r E l p 38 E 1 3 i r i s 3 c i r 3 i 3 1 ic u E 1 8 s r p if f F s r p s s i 2 1 l p y o E 2 1 py i i l i EF213 18 3 3 p 2 i s 2 1 3 y i i f ol EF i i y c i E F 2 y l f c o l EF 2 33 c i F 3 l 3 y l o i 3 EF2132 F 3 F l i if l l u F i iu E 1 i l l l i if F 1 l l 13 i E 1 i l i l i 1 1 olius l i E E i f i u s E i f o u 2 2 f o s 2 E f 2 2 o o s o l F2 s l F F F l iu F F l i

l i i us

E i u E E u E u E E s

s s s chamaephyte lineage (leading to the clade Lobelia physaloides - L. tomentosa) and a hemicryptophyte lineage

A

A

Y A

A F 3 a A Y

A Y 05 c 5062 A i AF 3 A Y A A A Y 3 r

A

AY3506 5 A Y Y3 3 Y 4 A Y a Y 3 6 s A 0 A F 0 s 62 F 3 i 9 F0 DQ 2 3 d 35 3 5 5 A A 6 Y 0 r 0 Y 62 a 5 4 5 0 4 7 5 hy is

4 3 i 54 e l F Y 7 bil p r 5 3 AF 3 0 1 c i 5 6 0 0 0 6 AF 9 3 t ilis b e o 0 1 6 49 0 3 5 7 u pi A 5 6 a ii t l 4 6 5 6 6 2 t 6 1 a s t A 938 1 5 4 a a ns 0 6 06 5 0 n 2 1 9 2 a AF F05 0 2 7 H 1 lu i 0 3 s a u i 05 F 4 a 4 6 a 7 co 0 23 6 n H 6 6 u in a 5 8 a a d 5 1 i n a 4 o q p et L i l 0 9 7 0 1 l e i g n 1 30 6 2 u 8 l r q n 4 L a 0 y A A 1 a y i gan u 1 o ig 495 e a 5 1 y e 54 1 3 1 L l 1 9 c el d er e i l 9 o 54 AF L 49 1 1 L p y

F0 F0 1 s a a c co 9 b b 5 1 o a i le a 1 L P i a a 4 L b d a b L a se a na y L Lo o an i i 1 er i e e a 9 ot o L L t a e g Ly b n g na a 96 1 5 o o n r si ra a 3 e o a lia ll i 1 1 ob b ri lia i 0 8 L m ba 5 5 s g a l (the ancestor to all the others). 51 t L ol y o a l A ro n A L ob e n i a i c L b L a m 9 1 om l l f gi b l b e e i y i e o ig 5 p r 4 4 a l n Ly 1 o a i s i ia a AF L be t F 1 sip L l i g a r o l n AF F 2 o a a o e a t y a ta u n e 1 e i i s b va 4 li g o e k i 9 e 9 ipo al we i a 1 1 l g n a a a 0 ob be o el l n b a o r 0 b p we i L b i a l ren d ak l 9 li l n o i gi a m c 1 61 1 L L s l e t 5 b n tu a a m i i wn n AF05 L i a a i i 6 o 54 i lia i 0 b i i y i 5 a ra r in L e o 0 om y e a k o g a 5 o l wn 6 y ip a i d a n P n n n A 2 s a x a a cal mc o i y s 5 L ia e 3 L Ho i e tr be w in 4 s m li g n g 5 g c l l l gus n o im y A 3 s 3 a o c i n u o 1 fi a

y i y i f i F e w s s a a n 96 v v i 49 ys om a f 1 g i l i 1 i 4 a 9 a H o 4 s l a n c m ipom l i a y 1 t or 4 p ia e f w F s i g s 1 i i i e p D D o n i 0 a ol a u cr n g a e m 4 a o 5 n ia 9 3 i d a n a w i an v a m D i a m i o ca 3 o p n l i 0 L u a olo 8 1 ati l 5 i x n h 9 i g i i 54 L p s ric e n tiss 4 a w ner i co 6 4 p o dif rmi ea 3 3 D o 6 1 o y a c a l 1 n i ss 5 i m la l a om e 6 a ys e i 4 sp o u l 0 4 s e A AF0 a y D wnin n g ia a 6 9 o a c n a 1 o c ti t el s 1 om a 3 5 i rn t 4 si ys s i a u 1 e 1 rdin e D a s 96 h e s 1 1 b 4 w rt p o l 3 b g n na o F 46 m a a e 8 i i xif n a i s s a oli b t l A 1 i n 06 4 2 n s b 1 D r ta i i 9 1 c a g F mont 0 a Lys p a tra a b g wn l a s o 3 4 n p hi sa a F m rat o l 2 g i d 0 i Ly t n n ip i l a i r 1 D i o 5 48 o ic 3 4 i a F05 ia us s o 7 1 L ia o a g hi a 2 w a o r no o o

AY350632 1 Lobelia tupa r L A emat 6 3 5 Ly o l L 1 1 n g o 549 L e s a 4 c A e i 1 m l or L 6 3 nat p da ta a L o ys m o a i a l 4 7 1 Do o i 49 s m l a L 1 6 n a yl p i B ya 3 0 D gi vi t is a n i i s u A ip b le p n D 1 6 1 wn AF05 1 s ip m v a i l Tr 3 7 p dat L a a a 1 4 D n a or i i c is C F 338 3 n g ata n F054949 4 ip rac a c i tre i C 1 24 1 o w i i a A 4 55 ip om y ndr a h r d r n F 6 3 Downin r o i r a AY35 16 3 0 cu d 95 y L d A 6 1 n a 3 1 L a g a 4 o w in g i o F o si m 1 r A 1 3 D AF s y o m a 6 0 9 ta B l in e ii AF 1 3 42 o n n a cus p a 4 ola w i gi dat ic 0 ip s m m p h a b ta AF F 6 3 0 1 D i i 7 1 i oc a i 1 9 1 s ta 5 4 a i AF F 6 o n n b 0 1 ip omi a y es i ri a 1 1 4 0 D w i ia p da c 496 9 Lys om i ia li n A F 63 6 1 u i i 5 a s c 9 A 1 4 84 D o n ng a o s n a t F 7 3 1 w a cuspi p r p t 5 y L sp ip n a A 1 a A 49 p p i F 6 3 91 0 D o ni ng l a c A A 0 sipo m s h e r n i A 1 1 w i a c cus l a ys ia s a i i F 33 3 3 3 i i t v r F F 5 ipom p ut h t at pa a A 6 o w us a pi F 64 1 1 Lysipom i e o F 1 D n ngi s l a a A 0 0 1 a n p on A 6 3 1 6 0 g a c il A 0 Ly ipo c c A D o w u ta v r 5 mu n ov s a 1 6 9 3 6 5 1 ni n s id F1 54 5 1 m p u io AF1 i gi a p u a F 49 L h a 1 2 3 3 1 D o i u p 4 ov m i AF1 1 n n v A 0 9 y s i m i n ison s a 2 6 3 D ow a p s rn 6 94 L a a g AF F 35 1 1 w F1 5 4 5 s i 8 a 1 ng u o a 3 y p s ia d s il A F 1 6 3 4 1 o gi a A 4 4 2 3 ip ov 8 a 2 ni i c c rnuta 9 0 s om c 1163 A F 6 3 in i Y 3 2 om p l oi 69 ii 4 1 D D owni g o nut 5 1 1 1 ip obu A F1 6 w n ia b c 3 4 9 ha 336 35 1 in i 6 o ia m i eli 8 de A F1 1 1 o g a or 22 8 1 Ly L L a w i b c A 63 1 L y y ia m g 743 A F 16 63 71 8 1 D o in F1 AF c laris 34 wn g la 07 G s s s i m n s A F 1 6 n n ia bi l ipomi i i s a a 6 4 1 D D i g e 3 1 r ys p p p o u oph t A F 3 w n a la A 134 3 a o o a u 633 3 1 Do n b la 4 A F Pl m nti lti A F1 1 w el l 8 1 ip m mi n r la 1 1 ni ia e 5 F 1 e flor ila A F Do o ngi b a 3 862 1 Pl at m o i o t o 16 6336 5 g h ll 9 1 m a sp a a lac i id A F 3 9 1 D n bella lc A 3 62 a yco a v o 63347 1 ow i he 1 48 t i s id es a A F1 0 1 n gia a u C F 3 ty o a u 8 1 63 4 D owni n i p 1 7 c d t g bp 7 e A F 3 2 1 w o 6 Codonop h nov inia 1 6 35 D o ni a d 3 1 1 o o e la 2 s A F 3 6 ing i ta E E o 4 d n c e 8 1 3 1 D n g e F 8 1 C o n A 6 3 57 n a pulc a E F1 n 6 n g g a dul lta ta 1 3 1 w i gi la t a 1 F o 0 C o r 1 AF F 6 Dow n e a l 9 1 9 p od d a b t 1 3 6 in ia la t l 0 0 s 1 o ndi e 28 a A 6 6 1 w n g e e 4 9 i n ra r if F Do o n a a h la 6 F 04 4 s C o g er A 63356 7 1 D i i l lc l 1 F J 6 p n o s n f i F1 1 2 90 ow n J 9 6 0 1 od o p is d lo a a A 6 3 8 1 ng ia u he 1 9 8 2 ilo p s j if n 3 9 w i g p lc is C 8 0 1 s ono s is av lo rum a AF 63 7 o n n u n a o 0 3 C u i ne ru Y 1 6 D w i ia ig is t d 3 1 Co o l s an A 633 3 3 1 D o n g p n cornutau o 1 5 d a p t m F 3 1 D w in ia ns g bi rn DQ FJ n 4 1 d o v s an r ica A 6 52 9 n i i o o o n a is v F1 9 1 ng a ic 8 5 p 1 C n gs o A 1 09 Do w i ar b A 10 72048 s C o o r pi s F 3 0 1 o ni ins lla Y is d o p m h a A 0 3 8 D ia e a v r 3 o p s lo e H 6 3 4 ow g b la t a A 2 274 p d on s is o su 1 3 9 1 ningn l v H00 2 i o is d n A F 6 3 ia nu a 0 1 1 lo n op t p e l 1 3 4 1 D ow ni g be or t A 4 s o a ilo s a A 6 7 D w in a c u F 8 7 C C u p s n t F 3 1 o n i i rn 1 2 1 o la s is g sul a A 1 3 4 1 g b o 8 18 od d is p s 6 3 9 in a ic a a AF 3 Co on ono va t i h a AF 1 3 6 1 D ow n gi b ill t 4 1 r a lo e 6 D w n a s da There is great life form variation in the Lobelioideae at the 4 n n u 1 5 C d o p m g s AF 1 33 1 o ni i p A 8 o ono p s s u F 6 409 3 D w ng spi DQ AY H 34 1 d s is o h la A 4 1 o i ia u a 00 4 C o p is p d e F1 63 3 D n g c ill 8 5 8 3 o n s k i e n A 3 5 w in a s 89 AY 48 2 1 d o is k a lo st F1 6 6 o gi u 45 1 C ono psis w su a A 1 33 2 1 D wn n p illa 3 195 7 o a a F 1 o i ia us 9 22 1 d ps wak k la A 16 9 D g p lla ta 1 04 1 C o la am F 339 3 1 own in a a C A C o n is nc A 16 3 D i usi id o Y 8 do o lan a ii F 3 5 wn ng p a do 3 1 od ps eo m 6 41 1 o i a sp at n 22 C o no is ceo lat ii A 1 3 5 D n gi d A op 0 od n p t AF 6 34 1 ow in a cu la EF Y si 66 on op s ub l a 1 3 6 D gi u 3 s s is ul ata F 6 1 wn cuspi s 20 2 jav 1 o is tu o A 40 o in ia o la 67 204 Le p lanceolatb s F1 D wn arn u an p sis u a A 163 404 1 o ing c os 01 5 ic to l lo F 7 D n la rn A A AY 1 1 a co an sa A 163 41 1 ow el a a F Y 3 C C su d c 3 8 D c os 04 32 2 a an b o e AF 6 5 ter lla m s 7 2 20 m a sp n ol a 1 33 or re li ili a 92 04 50 pa rina gr at AF 6 48 1 P te re sim or 4 6 1 n ja ac a 1 3 1 or e uat is i 1 1 C um c po il F 3 7 n aq at ev lor AF C y o ana n is A 43 1 P ge a rn na br co 0 od an e ica 2 e li o ta ar n 47 o a a l rie AF16 63 7 L el ia n v co 9 no nt a n F1 33 1 ow o lor r 24 ps hu nc sis A 6 53 H ning m o H is s l if 1 w gia nc va e d o oli AF 633 1 1 o in co lor lia ic ba a 1 43 D n co intergeneric, intrageneric and intraspecific levels (Figure nt en tu F 3 1 ow gia n hu tr s A 16 12 in co s ifo 1 D n gia a pii an lia AF 634 8 ow in in lu n F1 37 1 D n y iga uu A 63 ow ac s F1 396 D ningia b ns A 1 w gia ga 163 02 Do in ele AF 34 1 wn ia 16 88 Do ing legans AF 3 1 n e 163 32 ow a na AF 91 D ningi yi 22 6 1 ow a a F 38 D ningi in A 63 0 1 w ia y F1 14 Do ng A 29 1 ni ina F2 1 ow gia y A 342 1 D nin na F16 38 ow ia yi A 91 g na ii 22 4 1 D wnin a yi lup FM AF 914 Do ingi ciga 21 2 1 wn ba 271 AF2 34 Do ia FM2 6 1 2291 1 ning na i 127 Cam AF 112 w ia yi lupi F 11 1 pa 29 1 Do ing iga M21 C nul F2 36 wn bac 7, Table 3). Only in a few cases is there a homogeneous A 271 amp a b 291 Do gia FM 5 1 an rous F2 6 1 nin a 212 Cam ula son A 342 ow in 714 pa brou eti 6 1 D ngia y FM 1 Ca nula sso ana AF1 382 wni yina 212 m bro net 63 Do ia osa 717 panu ussone iana AF1 2 1 ng a acte FM2 1 Ca la b 914 owni olios r br 127 mp rous tian F22 6 1 D ne f a va F 12 1 anu son a A 4 sio ntan M21 Cam la br etia 2291 1 Ja mo 271 pan ous na AF 826 ione osa F 3 1 C ula b sone 22 Jas rymb M212 amp rou tian DQ2 8 1 e co 720 1 anul sson a 82 sion a FM Ca a bro etia Q222 1 Ja upin 2127 mpan usso na D 827 ne s 18 1 ula br netia 222 Jasio pa FM Camp ous na DQ 29 1 e cris 2127 anul sone 228 asion iflora a 19 1 C a brou tiana DQ2 1 J essil arian F amp sson 567 one s sp m M212 anula etiana Q304 Jasi a sub 780 1 brous D 0 1 crisp ristis FM Camp sone 22283 sione bsp t 212726 anula tiana DQ 3 1 Ja spa su 1 Camp transt 2283 ne cri FM2 anu agana DQ2 Jasio chii 12781 1 la tran 2831 1 eldrei Campa stagan DQ22 ione h FM2127 nula trans a 34 1 Jas rennis life form correlating with a certain clade, as indicated by 22 1 tagana 228 e pe Campa DQ2 Jasion FM21 nula tran 568 1 s 2721 1 stagana Q304 ne laevi pa Campan D 1 Jasio bsp cris FM2127 ula transta 22845 spa su 23 1 Cam gana DQ2 asione cri FM panula tran 2832 1 J na 212724 1 C stagana DQ22 ne monta is ampanula 6 1 Jasio ar gracil FM2 transtaga DQ30456 ontana v 12725 1 Cam na Jasione m ana panula transta 222840 1 a var mont FM212708 gana DQ one montan 1 Campanula 835 1 Jasi ularia F specularioid DQ222 itima var sab M212709 1 Cam es Jasione mar panula specula DQ222844 1 ontana FM2 rioides montana var m 12710 1 Campanula s 2836 1 Jasione pecularioides DQ22 latifolia FM212707 1 Ca Jasione montana var mpanula specularioides DQ222839 1 ria e maritima var sabula FM212706 1 Campanula specularioides DQ222843 1 Jasion Jasione montana var latifolia FM212705 1 Campanula specularioides DQ222838 1 maritima AY322032 1 Campanula primulifolia DQ222842 1 Jasione maritima var the low Consistency Index of life form overall (0.27 on FM212704 1 Campanula lusitanica DQ222837 1 Jasione montana var latifolia ca DQ222841 1 Jasione m FM212698 1 Campanula lusitani ontana var gracilis ula lusitanica AY322060 1 Jasione laevis FM212702 1 Campan a AY322059 1 Jasione c 1 Campanula lusitanic rispa FM212700 AY3220 nula lusitanica 61 1 Jasione maritima 212699 1 Campa A FM usitanica Y322062 1 Jasi Campanula l one montana FM212697 1 tanica AY322063 1 panula lusi Jasione sessiliflo 1 1 Cam EF0905 ra FM21270 lusitanica 57 1 Jasione h Campanula AY32 eldreichii M212703 1 ezudoi 2080 1 Wa F panula cab E hlenbergia 8 1 Cam zudoi U177773 hederacea FM21272 ula cabe 1 Campanu Campan doi EU1777 la primul 12729 1 cabezu 75 1 Ca ifolia FM2 panula oi EU17 mpanula 1 1 Cam abezud 7772 1 C alata M21273 nula c EU ampanu F Campa udoi 177776 la pereg the tree shown in Figure 7). One example of local high 7 1 abez 1 C rina 21272 ula c i AY32 ampanu FM Campan ezudo 2055 1 la lactif 732 1 la cab EU Camp lora FM212 panu culus 17777 anula 1 Cam rapun AY 4 1 M lactiflo 12730 nula ana 322 ussch ra FM2 ampa berti 067 1 ia aure 46 1 C la lam lus AY32 Muss a 0905 panu uncu 2029 chia a EF Cam a rap AY3 1 Camp urea 609 1 anul tula 22049 an 304 amp la pa E 1 C ula pe DQ 8 1 C panu ina F090 rater regrin 1273 Cam egov A 559 1 ocapsa a FM2 39 1 herc ina Y32 Roe co 2127 nula gov 2074 lla c nges FM mpa erce a EF0 1 R iliata ta 1 Ca ula h ovin 9056 oella 616 pan rceg AY 0 1 cilia 304 am a he ana 322 Trac ta DQ 8 1 C nul cki D 078 1 heliu 461 a la be na Q22 Tr m ca Q30 Camp anu tinia D 282 ache erul D 17 1 mp jus ttii Q3 5 1 lium eum 046 1 Ca ula se 0457 Trac cae DQ3 19 n che ia DQ 0 heliu rule 046 ampa mar ifol 304 1 Tr m c um DQ3 1 C ula und EF 569 ach ae 613 pan rot don 09 1 T eliu rule 304 am ula co AY 054 ra m c um consistency is in the clade Lobelia oligophylla - L. arenaria DQ C an eno ri 3 0 1 che ae 12 1 mp st e 220 Cam lium rule 46 Ca ula chz ii EF 51 pa ca um Q30 5 1 pan cheu in 090 1 Ca nu erule D 61 am a s erm AY 52 m la pr um 304 C nul h ia 3220 8 1 pa op DQ 0 1 pa ula difol D Ca nul inqu 462 am an un ca Q3 16 mp a ru a 0 1 C rot i F 04 1 Ca anu me DQ3 14 Camp la itan M 57 m la lian 46 0 1 nu us atica 212 8 1 pan drab a 30 202 mpa ula l rv DQ 737 Ca u if DQ 2 Ca an la a lia 30 1 mpa la e olia AY3 1 mp nu difo EF 45 C nu rin a pa n sa 09 80 am l us 037 1 C m otu ito A 05 1 pan a dr 322 5 a la r sp lis Y3 38 Ca ul ab AY 202 1 C nu e gi E 22 1 mpa a ifol 32 0 pa ca fra lis F 00 C nu erin ia AY 201 ula gi 09 7 am l us 32 am an bsp fra EF 05 1 A pa a er Y 1 C p lis 09 43 z nul inu A 36 am is su sp gi E 0 1 orin a s 27 C gil ub a la F0 53 Ca a pi 21 1 1 fra s s fr A 905 1 1 m vi na M 62 la ili bsp phyl ii Y3 3 C pa da tzii F 04 u ag iso lin A 22 9 am nu lii 3 an fr is su a vo s Y 0 1 C p la DQ mp la l ul ca de 32 15 am anu pu a nu ragi n oi s AY 2 1 bic C pa f pa sp in e 3 02 C pa la a 7 1 m la am ub lat tin E 22 7 a nu fru lyx 2 nu C s e la ia F0 0 1 C m la tic (N11 in Figure 3), in which all exhibit the hemicrypto- 6 Ca a s e 5 p u 04 1 1 ili la a nis EF0 905 4 a an po lo Q3 26 amp 30 g u ul ia 2 1 F m ul ly sa D 6 C 6 fra pan n ce is E 90 2 e pa a cla 04 1 04 la m pa la en is F0 53 1 eri nu ed da Q3 28 Q3 nu a m nu c al D 9 0 1 C a la ul D 46 D pa C pa la id s Q 05 am a m is 0 m 1 Ca m u m ali A 3 27 C pa ng o Q3 a 25 1 a an ra id Y 04 a us llis D C 6 24 C p py ora A 3 5 1 C m nu ti 1 04 1 ram ifl r Y3 22 79 pa la fol 29 046 3 am la d lo 05 1 am nu ba ia 6 Q3 3 2 1 C nu py n E 2 3 p l 04 D 46 a la cu ico a F0 205 Ca a la fo 3 DQ 30 22 p rs tic A 9 1 E m nu fil ur Q 6 se a a F 0 2 d p l ic ii D DQ 04 Cam panu la ve p E 18 55 1 r a a au 3 m u la ian a F090 34 6 E ai nu di lis Q 1 a an u car in n E 1 d an la ch D 34 C an la as ia F 39 Ed rai thu d ot 0 1 mp p u in lla A 0 55 1 an ic o 22 m an m u Y 9 E raian s ho m 3 06 Ca p ste p A 3 05 1 th p a Y 46 1 Ca tom ld la neri 2 3 1 d u um to A 8 1 am a u i a Y 2 7 C rai t s g i m 30 ul wa n ra n i A 32 0 1 am a hu r lio a Q 60 07 a a i Y 2 1 nt s a D 4 6 1 C la p ys A 3 0 1 C p h te m 30 4 3 pan u m ul ettia o i Y 2 2 1 a a u ini 1 an a an or z ki EF 3 2 6 C m nu s nui f Q 30 20 am p p c ii 22 0 1 a p g o D Q 2 C C m la ie k E 0 43 Cam m a la ram fo lius D 3 m 1 m a u d c F 9 038 p nu s liu Y 1 5 a ul n a ie s E 0 0 1 a la pec s A Ca 0 C n a l n i 9 5 C n in 11 1 6 1 a p u d e i F 0 34 1 a p u m i if 6 0 04 p m n la b AY320 5 C m an la e o ol phyte habit, at least facultatively. Although the overall low 4 1 4 a a u m a 905 d sa ius 0 6 3 0 m p n u n A 4 1 am p u b iu 4 6 C m a c a F0 9 C a la a m Q3 0 DQ 04 Ca 1 a p e diecki A 2 52 1 a p n rb D 3 3 1 3 d la s ica Y 9 03 C m anul u m a 0 C m in n a A 0 1 la tr ira t 2 6 1 a la nu k a ic F 3 7 1 a p a DQ DQ 4 3 C u w rg n a AH 2 2 1 C m a a b 3 n pa a ic 1 2 0 a p n s ide il 0460 30 7 1 a ha n n a AH 8 03 Campm a u is 3 2 4 p m ga r 00 3 1 n la ar nta 1 3 a la a ga an a EF 4 3 p u i ma Q DQ 2 7 m u l ca r y in 00 821 3 1 Ca a l n ta D 2 C u k t a E 5 n a c M 1 Ca 1 a ga s a c 0 6 Ca mpa a ula s ur ti F 2 5 an an la r e i a F 9055 45 1 co v c 1 3 p p u a r n n E 0 3 C nul a a M 6 ha e F 9 1 m s p F m m n ul la ll ia ta E 0 4 am n a p t a 2 27 a a a sc a a F0 05 C pa u ri 5 p u ll ca E 9 5 1 l c ri 0 21 C pan n h lag e a F 0 4 1 C a pa n a e ta a 9 1 C m po a p ia t E 9 5 1 m u pu tra 1 a p e tr r F 0 0 2 C a nul l F0 FM 2 am la c ch s lla is A 9 5 1 C m p a E 19 3 C m s e e s Y 0 0 4 am a pu n e 5 1 C nu a a n ist r e E 9 5 3 p nu a ct a 20 l en t n 3 0 5 1 a a 0 8 1 C u e p s e m AF F0 2 5 am pa p nc 9 9 n rt s e r rm 5 3 1 C nu la u ta 32 0 6 1 f a o u m EF0 2 2 a t Y 5 9 mpa a po p n if t 0 9 0 1 C panu nu l p n a 4 5 a 9 s ub b a u e E 0 5 m a u c t A EF 0 9 a e s c t r 9 5 17 C a t a 3 4 C mp l b s fe o i a a A F 07 1 m p la p nc a 0 a u u a d c l m 0 9 4 a a unct t 3 1 45 t m i u u a EF Y 05 1 C t a Q C n s sp la sp 9 2 4 m p n la ptar a D Q 1 0 a la o p ic li s a D 3 a a u 3 1 a l b u c a s o o 2 0 2 1 Ca mp p p ta D p t e n f s a AY Q 0 5 3 a n la at 60 Q 7 a r a m a i m o t 9 2 6 1 C u tarmi m m ll a ri a a D 3 4 m n l b a D st p u orb o 05 0 8 1 Ca a a u a ic 04 59 re m e la c n at DQ Q3 3 0 4 m p n e Ca t ta su u um a m 2 la i t ifo consistency is partly biased by an uneven taxon sampling 4 ne a m u n n DQ 4 5 2 1 C a u n u s l e yt e m s i u m 2 m p c Q3 0 1 l a t b i p n li a D 04 5 4 C a n la s v li i li 3 e fe e n h u r 3 0 7 1 a ul u o f fo a var D 0 n y o is com pi o liu D Q 0 1 C C m p n ol tr C P hy ex a 3 2 2 a c bc lu l 0 la te x l if s A Q 4 5 mp p a u a i ia fe s 1 1 gl a fo flo um i E 3 04 4 n o c a DQ u As P y pl le l n i i e i F18344 5 7 1 a a l glo n a v e 5 ru c AY 3 04 u a r 46 la n 2 h o p u o n i d n F 6 1 am m n f p a n 9 1 1 a i m AY 0 5 7 C a l ra e ar c u a 7 P ma s r on u n o 0 u a it ta 30 e 5 o m o u AY32 4 57 7 5 1 C pa n me rt apit n p u m a i o h pa a a l a f 4 0 1 s l t EF 3 9 4 a g d Q a 92 84 la ul c a 5 1 Edr a m pa u a t pt m 0 2 e y m lim A 3 2 0 n u a la 5 5 ro r an a t a 7 3 m l lom D a 3 1 Phy h t u ap A 2 1 u a ka r a e u yt e c a t a Y3 2 5 7 C p nu la ata mp 6 32 7 e ro a n j i ic DQ30 0 1 l r l C n Q 04 1 P ma v r c a Y 2 0 3 1 C p a a s mi lat 2 0 a r ocar l d is E 9 am a ra a P pan t i r a A 2 0 1 an n c er D 3 1 et e e a lc r 3 7 3 C l t 1 Ph pa li a i A F 2 05 1 a i a a Ca AY eu m r p e 07 2 an ul le k c J4 22 70 P um m b li i E Y 2 6 S h a 4 Q 1 5 iv o i 0 2 1 a m pa u i F 1 n m u m e fa ni AY Y 2 S u t yr ro f mp 3 0 a l y A a t o 1 9 d a fa if n i F 3 2 00 1 y m r s D 8 9 1 y la la i A 9 5 t l a a 3 ne a c i en e 3 5 C p a tr ach l 2 5 Y 2 a a h c ifo A 4 0 y m h so c i 5 s u e Y3 0 2 0 1 S nul t a 8 6 y ne s a s i i v u n E 0 p a r 9 4 2 C i c i ra A Y 2 m u h A l a 3 2 5 2 1 Ca a l i C 5 4 0 A s a n s s i v ra A Y 9 54 8 a c y m u a e a t a A p a n a en 5 0 n i o F 2 1 s i 1 0 u r t s A 32 2 71 3 ym mp d t E Y 0 p c 4 2 1 a c a 0 h u t e h 4 3 58 m u o AY3 Y3 3 2 Sy n a t a A Y32 s rs te fl lo a Y 0 02 1 A an c E 2 i e r n umv e s a A h h a 0 2 1 t A Y 1 fo l p f a A 5 Ca 0 EU591967 1 Adenophora triphylla A ou D 3 y l 3 F s As l t i n F 2 5 ul i 4 g t 2 enuifo u s ti i c a 1 a A 8 u e a 9 m um s Y t e a ro l 3 2 0 y 3 3 a F0 i 9 t l s 8 Ca p a a F0 Q3 s 1 st g n 2 4 a h 0 u p o a l F 3 s F a tr a J 2 0 8 m oda ri l i Y 0905 1 C m l Q a DQ o r 22040 1 l 1 ca 0 1 a i 5 20 i p e ra 22 0 3 C s Y p 3 p h n n i Q3 7 i 7 y a 3 a e c a y e 22 20 D s e 5 a c 5 a la a yl s 0 0 6 m u l o 1 1 o 4 l i 3 5 1 n 5 Le ul 2 p r i o 3 9 ph y d a rs 8 mo l 3 2 a u A L y 9 a DQ 7 nensi n e h d C D m l 9 m m 4 n 7 2 8 Q u la u pt t h 9 0 Tr eg mo f 7 i 1 d 0 C u 2 2 i 1 n a p a f 2 5 p a r l 5 syne i 0 Ca n l 0 2 c a 0 C a m p i w p o p 0 0 012 7 1 o ar 2 a a n r e 8 n L u r e t 0 a 1 l 7 r D 9 o n t a div 20 1 p a y n l 4 1 a 0 0 1 u a s i A 1 re re so 4 0 C m a i i 9 5 3 o p i l v 7 7 a a 0 o rip n 1 l 7 m 0 n i p o 23 n u Q3 a 7 p d a i a p o a n d h h 0 5 1 C a l t 4 1 t 9 8 r 5 5 2 u b a 1 m a a p 9 p 0 M ho 4 a a s a h a F 1 6 a wa e 14 p a nd h e D a 3 3 p ra d j n u r o o 5 1 8343 1 Ca 6 i ra 8 Ca m p a m m 5 8 6 a m 9 1 m a n o 7 8 8 Ca m s 3 p p 0 o p i n h 1 a n E r 0 u l f fm DQ m s o or Ca p k 1 7 Q 6 0 o 1 i a 4 e 1 a a ra m m m a l 1 C s or a e c 4 a r S f 0 5 1 p o c u ra h u n l o o r 1 h u h 1 a 8 am r pa nu m m h r 1 1 a a o 1 C D p h m a 1 m h a 2 5 1 a n C C no a ym ul l s l a u h C p o a n p or 1 m o r a e AF Ca 30 22 a p A H al

am mp h r a 220 a G v a 2 01 0 p C i a n n b a arm n A f C ph ra t A a u h H am p b m o a 3 o r n l a na C 1 h Ha amp m a r 3 C p u n 9 o A e p a l n s 1 de de r m u n 045 am i n l d ego p o r an a i a n a d l d o u 1 no p h p (for example, there are only two species of Lysipomia sam- th a a o r n 1 a x i p o l a ii Y3 n te l b a i 3 DQ 1 d pa a u ar A l i L 1 e r n h o t i e o r a 4 e l e o p h e pa h c i n i AY 1 h a i A n n n AY A F0 0 9 e ho p a a o e n i e g C y o n l a a 4 n ul h d p h n o n r d n o p n y a a c a i 1 4 p p e u n 3 d a an pu nii 5 o a r i n E n n 0 1 1 h p nu u t 1 deno o o s ar e p h o a fo DQ A e n o o nu t o a 0 b a A o a o onie d l 0 b a AY322 4 A op c nu c s a 2 5 4 p o s l a a d n o p p n p p 590 2 2 d e i n a s u n h ni 2 n u n u o h i l 0 A is 2 6 p xi 0 r l 1 12 r e i e n h o d 1 h ho s e l 4 4 1 A d a s a 2 t s o e c 2 A e u l a s a i 3 0 3 en 1 A d la a oli 7 h a g d o h h e r f n 6 o 5 1 d u 3 a 0 A s d n d l a d o a r 2 0 3 1 r n Q 07 a o a a i e d r a e 0 r a 30 0 r s lo y m z ac c 1 r A belli d Y 1 y Y3 a if n b osn 2 3 A si 9 4 a a Ad r f AY 1 t r g i a 0 d p e m D 9 2 a A m 7 a ea c 5 o a fu Q e i 3 A 4 1 A m A 9 e p a s 09 1 d 2 s r e ti a 1 k 0 s h A 0 6 0 h l u 3 a a Y 1 1 na ii D F0 1 a s es l sa 2 t l nd c e F0 0700 6 p az e a 9 i n 0 0 p 7 id ow e di n s 2 s ri 8 8 l t 1 A A 3 s a r 07 9 1 i w AF 7 n i 0 a u E 2 7 o 9 i n d i 18 i 1 g 4 f i if

f a f i u Y 9 7 c a l 7 i 0 6 bs F 2 1 o c n an o o i ii 2 i ol 5 c s i t d a l 0 0 t F0 9 A 9 9 a 8 e l li a A i u r k 071 8 i AF i Y c

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F s sc A D 2 6 2 5 i Q2 i i s DQ28 DQ D Q o i i e 0 p 6 D 2 6 F 8 n Q f ora D D n i a r i ie l s ana s 4 Q 3 7 3 3 5 g t f 0 k Q g r a e o Q 2 Q i i n a a i D DQ 8 e b 2

s 5 1 2 2 c DQ 4 35 u n c 3 s b v 1 1 d ly 8 s e a 2 D 5 3 k u 6 2 6 u r Q 3 7 o t 2 a DQ 5 a n n

5 1 n u D 561 r 2 i f a 85 pet i a 6 1 8527 D i Q 1 3 D g a a 6 5 6 ea s

2 D 6 r u 35 27 a 3 28 7 k Q 1 par l a 1 A n k n 6 2 1 a a w e a 8 1 e o l 3 1 a a b 27 Q D i ia D 8 i l is 7 i i i 2 a o s 0 6 3 t a a p c l 5 2 l a 3 t a 5 1 a r 61 5 i

2 i 526 i Q2 2 Q 85 Lob k o o mi til S s m 2 6 1 s n he 2 1 8 lum 1 e m te s 5 6 m n o l 1 a i 1 7 l i a i D 7 k l u 8 L s i s nt 1 a o 7 a 2 5 c t f ir ia

1 2 a o e a T 1 e Lo a e c 268 1 e 5 e i 1 o o h v 0 Q L 1 l 85 8 2 h p h 2 5 1 h t onti a uh c a L e n L m r 6 h n a a es 26 a a m e b g 5 6 o C E DQ e 6 g r e a k s T L 1 Lo g x lla 1 2 ob i a r m pa a r s Lobel i be i a a l 1 i e g sa

l ylla e 28 r 2 9 b o a l m i o r n e a e y flu F1 o r r r on E T 8 p y r y e u p 5 1 a y le n e a d e e L l h l h t 6 el h er 1 t b n e h is B a a b i 5 e B erm u c 1 B s F a l h lo n ri p 2 1 L C r m Lo a l a a p b m 2 C C t ce r y 7 y D i is l e n p 4 o C y a 2 n e a E em L G e ia a l li n s 1 8527 t L o e y a b y a r s 1 Cl i x i e t 1 n t l ol 1 l i 1 l 1 Q a b a b 1 9 e l i ex a i ly 1 1 1 er C y e l e a c 41 Lobe s i a r U o b c 1 s ane i d i u n l d n a l 0 o i g ya a o i t b a el l e e i o 7 c ub D 3 a o c 7 o c a 6 C 3 a b i o b e a 4 1 C y xi a d 9 1 il C c o 7 a st o Cy l a L 9 2 p l b i f 1 3 i e s s e 8 Cl i a 0 l n 5 a b h i c C p Q3 56 03 s 5 1 o ar 7 lob e li 6 i a x i b i o e il 1 c 1 1 s to o g c s 1 C e l 1 6 5 e e i l a j ob b l a o g 5 0 G i 2 1 C l elia en t s 4 a 2 l i a 8 L el a 0 o 6 ra l x n 1 3 p o p 1 60 b ia is 1 be i ig 0 1 b n ia l 2 e C a 6 u L o ia a m 2 L f t ibbero 2 l 1 0 1 L l ia h bb tu 5 1 1 0 l yuc y 5 2 b l ax xi u il 07 Q a 3 o t 2 e 561 e s e DQ0 t el i i r 6 2 9 2 1 o b a 7 5 e t m 2 L e 3 o i a 5 9 D 1 a m 2 b r au 8 9 1 i o 0 6 h a 8 1 1 o e m a l b s 4 K n r DQ r l ni a 5 9 2 o y l 2 4 o a a 8 4 b n t 4 b rp 7 ia g i y o 8 6 L 2 2 er m yp r m 6 b o m 9 m 2 6 L e a o nif n a 1 00 a 0 4 s o po p c a 3 85 5 9 t a K 2 u 1 D o e ih u i u 0 85288 5 o vi 0 AY c l s 2 52 2 1 L o m rnh 62 n 5 o 2 l f a t 86 lor i ti e Q 1 o t F 2 52 2 t EB K a o w Q i a o a 8 1 u 0 a m m 4 6 L k li c o a s beli F 2 0 a 2 o Q 878 a o a 8 5 L a l 07 EU o o Q 14 a a r S g a leu a 8 5 7 1 s o f 2 7 S crost a c a DQ285264 1 Delissea subcordata 2 8 61 2 A 1 0 EB K n l D w l i i a D Q 8 I om a p 0 A 2 6 3 a u li i 6 5 1 ia e d n s D Q 0 s oto t a p 1 o k D 6 0 A 5 u o a Q3 2 8 1 o i 4 DQ 6 I ti i a EB B 2 5 4 1 ole n c v a L1 2 5 l K n e D 7 L a n a u e a Q 7 o 0 F0 2 0 le m a i 4 D 2 5 46 4 Is s E 51 i d c Q 1 a E 1 g ing r n D 3 60 3 1 s e B 2 85 2 u l c s DQ Q 46 2 r m sp K s o o e l 3 0 a i F n i boninensig e a D Q 2 5 b i 3 a o s D 1 I o E 4 n a s a a Q 7 5 l sp 4 b o n D 17 6 3 t a p K example, [21,23]). 5 3 1 L o u s 74 1 7 P o o ie s is D Q 1 e i m a 2 4 b i ta ch s 1 8 4 l s EB 9 1 obe e p tie n D 8 8 o a B r 6 7 L p a a 6 L b i ri 1 l n t i D F 2 7 2 74 1 1 e l E 05 0 i s a i c s 5 8 sp E fo a 59 L o s y i EF F1 Is o n D a d is rr s s 1 5 0 1 e ia p i l G F o b i ci n h E Q 3 6 D 1 Lo s s E 6 ob b s n Q 5 41 li s y i F 2 8 16 1 ia Q b e l gal e s D 7 l do n Q D 1 a c m Q 688 1 L o a sp e 9 L e or a i z s E 3 bel r mu o 3 2 1 b l g Q 7 1 L L e li o 3 Q3 05 ob ia D 2 6 1 d n 5 4 L lia u ni i Q2 7 7 3 b a a m 5 eli t i u s i D 356 5 Lo i l cr o E 6 o ar m r n 4 9 8 1 o l u n 6 86 4 L e s e p s D 3 4 be e d 1 5 be a c u Q 9 9 L e n cro o 1 F 2 o ip d a n i DQ 9 8 1 1 s o 4 6 li ta i b a r d 4 1 3 b a a D Q 6 9 9 9 8 1 Lo p a ma 2 1 1 L l c h in nna t o c o on D 0 41 5 e i r ia D F 9 9 9 ti a m r 6 a c a d il a 9 82 0 1 L i a d Q 1 1 1 li r it E F 9 9 9 Hy c o D 024 6 a ca a d i l 9 8 ra el a m a r es L o n ic i E 9 9 9 1 li i Q 2 H 1 Lo ca r i a s 9 9 b c i d 7 D 8 ob b din n a EF F 8 1 P e a m h i DQ i r li 9 9 97 o li a o es 0 Q 2 5 p L e d al E F9 9 7 9 1 b g d 0 68 2 p 1 L e be l rd s 9 L o e ia m u e i 3 o ia in a is E F 9 5 4 l 8 ob l b 99 99 1 1 L b e a o o 2 D 5 3 8 ia li i in a li E F 6 o i na e K 7 61 5 4 ob e a s 9 9 6 6 b l r 0 D Q 73 1 ro l nf a lis E 1 1 L a B D 6 a i i 9 5 6 2 o lia lin i Q Q 3 39 1 m e s a nf l lis EF F 3 4 1 L be nn i 20 5 4 1 L li s k at 5 7 3 o a E h D 35 3 6 o a a l E 3 4 1 1 L li li K D 1 1 L u al at a 9 7 g A Q 5 1 1 b k r DQ Q 4 3 ia u Q 3 614 6 2 L o e lo g m a 6 7 8 1 be l sp o M 3 1 2 b n r e D 9 4 2 Lobeo e a r EB ii n 56 4 o L lia a i F 6 7 5 1 L b li 234945 4 1 1 belia ob eli g u n i E 9 a p h o 61 12 3 v i s s F 6 4 72 9 1 o li s g d n L a vi fl E 9 3 L be e u o o 3 1 1 o e iv o s 6 3 o o r d 8 Lo L b lia a r i EF 94 1 lia r c o n 8 1 m v a F 6 2 L ob e a r o D 1 o e ld E 947 7 L ia c Q 1 D L b b li a s aldii F 4 22 1 l m a od ob e eli a tric ii E 6 1 1 ob e r on 3 W i li a rt F 9 2 L b lia m c d 5 as eli a xal a a E 6 7 5 o e DQ 61 im g go ta F 4 6 1 L a ro ta a la u E 6947 1 b li ma don n 24 merel tea m xi a n F 6 1 o e a ro o D 35 ir a n E 69 5 19 L b mac c d Q DQ L L 1 as pe flo a F 1 o a o n 3 6129 13 01 E 38 L beli lia r o 5 3 9 L la ue ns ra Q3 947 7 6 1 o m c d 7 6 E 56 3 9 o p ic D 6 4 3 L a a ro 0 1 0 31 bel r n is 7 0 li m c 0 s 1 F1 130 1 L y an s EF 4 3 1 e a odon 2 6 4 1 i g is F69 9 7 lia m r en 1 1 obel L 2 a a m th E 6 4 1 Lobeob e c K g A A Gr 02 1 o L ae a 9 727 4 L b lia B ful J3 M Lo b o nc EF 6 1 o e ma E E 2 a 9 i e b a 94 5 L b a p us E 1 E F 3 m 1 b a lia e e EF 6 3 1 o i s l a ta la U 8 F 1 4 m e gr lia ps F 94727 L el py i 7 99 1 410 9 Lo lia e E 4 9 ia mil ida h 1 2 4 4 a be a rin sp 2 1 el m u p op 3 1 1 2 1 to d ni EF6 7 2 b p n ila 37 A 02 8 1 t l e ti u 4 3 1 Lobo oca a us h 2 P F 5 1 M he ia p c s F69 9 7 L h c p 1 se 0 1 c a re ol E 6 4 1 p omi no P u 42 Lo on a q s a F 9 733 i sphag g is s d 66 Lo b o b ua s E 6 4 0 S sip omia a in e on be e p e a F 2 1 y p ia h ff x L ud e 5 lia s r ti E 69 47 L i m p a nis ly 1 o m 1 lia is gi ca 9 32 1 ys s s ffi a sis 87 n a P to d a EF 0 8 L ipo ia lu a ic n 9 e c r cor m e na 0 1 s py s ev re 2 m la at e bil EF6 141 6 3 y om lu r o s 1 ac d ia on n i F 4 3 ip am y b d nsi C la us b o to s E 7 1 1 L ys c p us a re y du o o p s 1 6 2 o yl cu o pho p rne if a 5 13 1 L ph cam e d L1 L s p oli EF 3 6 i o mp s ua us 8 E 1 ca op os e a Q 5 09 S h a ylu c te Evolution of the giant lobelioids p L187 79 U 8796 r p it ns D 3 6 1 oc s e n s 0 7 pu o ifo is 4 0 Si h mp lu a u 9 1 13 s si li DQ 62 1 ip ca y gig te is 4 S 3 1 ri tif u F17 4 5 S o p s an rm L 1 ty 71 C ge ol s E 7 15 1 h am lu ig fo 18 P lid 1 yp s ius F1 6 6 ip c py g e 7 ent iu hi c E 5 S o m us pa s 98 aph m Cyp a en 35 61 1 ph a yl tu u 1 g e s Q 5 6 Si oc p an Sp r r hi la D 3 1 h m lus ro s h ag am a ta Q 46 7 1 ip ca y di e m in el D 7 1 S ho p o no a if at 1 46 1 ip am n s roanu lis H c el ol a EF 7 S oc o di ria e lea lip iu 1 621 1 h og so to lia z tic m EF 74 4 ip op ua n ey u 1 15 S tr eq s s us thu la m F 6 1 en ogon a utu lu los s ni E 35 15 C on rn pa nu an ca 46 1 og co se ra n DQ 7 24 Centrop p n g uu F1 1 tro go amo us sp s E 746 3 en po n g an ub F1 C tro go z s s E 7462 1 en o ae su ns 1 22 C p n b ulo uta EF 46 1 ntro go an s n 7 58 Ce gr nu sp F1 61 1 tropo on za ub E 35 7 en og ae s s 15 C op b osu DQ 56 ntr gon nul Q3 2 1 e po a olius D 61 1 C tro gr nif 174 11 en gon ola s The nanophanerophyte habit (Table 2) developed early in o iu EF 46 1 C p n s ifol 17 52 ntro go lan EF 61 Ce opo so 35 0 1 ntr on DQ 61 Ce og egia 74 1 trop ost F1 13 en lept ri F E 6 1 C ea ste J58 F174 4 an for us 7245 E 461 Cy heca os 1 17 1 ot com FJ5 Ca EF 5289 cler gon us 872 mpa 28 1 S opo lute F 70 1 nula DQ 287 entr gon filis J587 Ca be 285 C po bri 254 mp tul DQ 30 1 ntro gla FJ58 1 Ca anu ifolia 46 Ce gon sa 724 m la r F17 1 1 ropo edu 2 1 C pan adul E 615 nt on m FJ58 am ula a Q35 1 Ce pog etae 725 pan folio D 27 ntro rgar us FJ587 9 1 C ula a sa 1746 Ce ma tinian 28 am rme EF 28 1 hia wes 0 1 C pan na 746 peta ylus ens FJ am ula F1 6 1 A p and 5872 panu lana E 528 hocam us sc F 40 1 la s ta Q28 Sip yl J587 Cam peci D 53 1 ocamp s F 281 panul osa 3561 Siph nsi J5872 1 Ca a af DQ 0 1 organe 84 1 mpan finis 5615 belia a the history of the Lobelioideae, most probably several EU Cam ula Q3 Lo olat 6437 panu spica D 1 peti mis 10 1 la su ta 285279 obelia lviifor Camp bcapit DQ 0 1 L on sa FJ587 anula ata 8528 opog es 277 1 ptar DQ2 Centr richod Cam micifo 29 1 ogon t ensis FJ58 panula lia 746 ntrop ganat AY6 7283 1 scopa EF1 9 1 Ce on llan 5515 Camp ria 35614 tropog ntatus 0 1 Edr anula s DQ 1 Cen dide EU7 aianthu tricta 4626 on gran 13380 1 s grami EF17 entropog us Edraian nifolius 5 1 C dissect EU64 thus gr F17462 pogon 3705 1 E aminifoli E Centro annii draian us 41026 1 tessm F thus gram EF1 ntropogon J587249 inifoliu e ans 1 Campa s EF174619 1 C gon nigric 9 EU6437 nula confe Centropo G 90227 23 1 Camp rta 174631 1 n sp UCB E anula thyrs EF entropogo U713346 1 Mich oides 4618 1 C rrezii auxia tchih EF17 ogon gutie EU7 atchewii 8 1 Centrop 13383 1 Symphy AF04265 cculenta andra armena urmeistera su EU71 F174662 1 B 3382 1 Campan E ra multiflora ula lanata 636 1 Burmeiste times (at nodes N4, N10 and N12 in Figure 3; with 81.6%, 4 FJ587269 1 Campan EF17 ula quercetorum urmeistera lutosa EU EF174634 1 B 713350 1 Campanula pelviformis era multiflora EF174637 1 Burmeist EU713352 1 Campanula tubulosa di EF174661 1 Burmeistera smarag EU713368 1 Campanula carpatha EF174633 1 Burmeistera domingensis EU713351 1 Campanula laciniata DQ356148 1 Burmeistera domingensis 1 Campanula karakuschensis FJ587258 EF174658 1 Burmeistera cylindrocarpa nula involucrata FJ587257 1 Campa EF174652 1 Burmeistera borjensis saxatilis U713349 1 Campanula EF174659 1 E trae Burmeistera crassifolia panula hierape E EU713395 1 Cam F174651 1 Burmeiste tchewii ra oyacachensis ichauxia tchiha EF174650 EU643720 1 M a 1 Burmeistera au panula incurv EF1 riculata 87256 1 Cam 74647 1 Burm FJ5 trachelium E eistera holm n ampanula F174649 1 B ielsenii 356118 1 C helium urmeistera tr 69.1% and 97.2% of all reconstructions with uniquely DQ ula trac EF17 uncata Campan ia 4648 1 Bur 587285 1 alliariifol E meistera ru FJ panula F174660 brosepala 41 1 Cam icha 1 Burmeis FJ5872 clerotr EF174657 1 tera resu mpanula s lia Bur pinata sub 6 1 Ca la latifo EF17 meistera sp resup FJ58727 ampanu a 4663 1 cylindro inata 260 1 C nctat EF Burme carpa FJ587 anula pu 174664 istera su Camp tifolia E 1 Burm cculent 25 1 nula la F174656 eistera a EU6437 ampa llina 1 B succule 78 1 C ula co EF17 urmeis nta 133 mpan 4653 tera glab EU7 1 Ca sonis EF1 1 Burm rata 7248 hamis 74654 eiste FJ58 nula c atica E 1 B ra refr ampa sarm F174 urme acta 4 1 C nula lia 646 istera 4372 mpa EF1 1 Burme refra EU6 1 Ca nii 7464 is cta 3386 mpanula latifoman EF 3 1 B tera s EU71 1 Ca hof ii 1746 urme odiro 1027 ndra ann EF 42 1 istera ana F14 hya hofm 174 Burm sodi E Symp dra ilis 644 eist roana 7 1 yan irab EF1 1 B era s 1337 ph la m s 7465 urme odir EU7 1 Sym anu oide EF 5 1 iste oana best states, respectively), or, less likely, just once (N3; p l 1 Bu ra 5159 Cam uncu is 746 rm sod Y65 4 1 rap iens DQ 45 1 eister iroan A 338 ula non i 285 Bu a re a U71 pan bo eimi EF 281 rme fra E Cam nula ssh 17 1 B ister cta 2 1 pa gro irica D 4638 urm a so 339 am la sib Q3 1 eist diro U71 1 C panu la la 561 Bur era ana E 381 am anu du EF 47 mei crisp 713 1 C p pen 174 1 B ster ilo EU 93 Cam a olia EF 640 urm a cyc ba 133 1 andr lidif 17 1 eis lo U7 279 phy bel ga E 463 Bur tera stigma E 587 ym ula ifra F1 5 me cy FJ 1 S n eri 746 1 B iste clo ta 85 mpa a sax ch EF1 39 urmei ra stigma 33 Ca anul au lia 74 1 B s cris U71 1 p ula ifo EF1 64 urm tera pilo ta E 44 Cam an llid 7 1 1 e ce ba 72 1 mp be lia A 46 Bu iste ra FJ58 74 Ca la riifo Y6 32 rm ra toc 872 1 nu llia sa E 551 1 Bu eis cris arp J5 48 pa a o U7 56 r tera piloba a F 33 am ula am m 13 1 me c 1 C an la r iu EU 35 Pl ist ris EU7 7 1 p u 71 8 aty era pilo 34 am pan med ium DQ 33 1 co sp ba 13 C ula ed 3 53 Pla do M U7 6 1 am an ua E 56 1 tyco n g u E 37 1 C p la m inq U6 115 1 C Ca d ra chh 713 61 am nu p la E 4 na on nd al U 38 C a pro ty U 370 rin g iflo a 1 45.7% of all reconstructions; Figure 7, Table 3). Present E 1 1 s 7 a a ra ru 4 L 1 amp ula o ta E 13 9 1 n c nd m 2 26 C n acr ec U7 35 C ari an iflo 87 1 pa m is a A 1 7 an na ari ru J5 79 m la em ian Y 33 1 C a can en m F 33 a nu s el 655 59 o rin arien sis 71 1 C a m ana EU7 14 1 do a ca U 67 mp nula ru eli ii C n n s E 2 a pa la in AY 13 9 1 ya ops a is 87 1 C nu rum qu 6 355 C na is rie 2 Cam a la ac us E 551 yan nt d nsi FJ5 72 1 mp j in U7 1 hu ic s 43 a anu la r lia E 1 48 Co ant s en 6 78 C nu a e fo U7 33 1 d hus lob tri EU 1 l bi ii E 1 5 C on a fol 5872 91 Camp pa nu ra rg U 33 6 od o lo tus ia FJ 33 1 am pa d bu ii L 7 8 1 o ps ba 1 m la rg 18 133 9 Co no is tus 7 1 C a u utz u s 79 1 d p la EU 3726 8 an re zb u L18 60 L ono si nc 44 1 C p c ut rin i 7 1 1 ept s e 64 3 34 a e zi A 7 C oc p vi olat EU 71 am ul cre a t J41 95 C o sis rid U 37 C an a ul na ii AM 1 odon do od v is a E 64 1 p ul n pi tz 9 o ir U 2 m n a a L 2 6 Cy no n g id E 25 a a p la in llis 1 3 98 ana o p is 7 C p m u p o s 8 4 ps si ra 8 1 m a an la m lli E 7 93 1 i s ci J5 0 a C p u o U71 91 8 C nt s ka lis F 3 C 1 m n a AY6 1 1 yph hu ov wa 37 1 8 pa nula 3 N C s a 97 9 Ca a la m tom s E 4 y ia lob ta ka 64 3 33 1 am u o li U 55 11 e p r m U 3 1 5 mp h du i E 7 1 m h o a ii E 1 7 6 C a pan ic e ali U 1 5 1 a ia ge tu day lineages inhabiting the Hawaiian Islands, French 7 2 1 3 M cl b 7 6 C m d d E 6 4 4 a rs s EU EU 8 9 1 a ula vi alii U 4 2 1 u d ul ii J5 3 5 C ula n id a EU 6 37 5 1 Mu ss u bo F 3 7 1 n ina v li 4 0 c s sa 71 3 pa pa r a fo E 64 3 3 s h ra U 13 1 m o ti tha U7 7 1 Ca s ia au m E 7 m z rin s n ii E 37191 ch o U a Ca A o u a r U 1 8 C m i s C z g h E 3 1 a p a r is E 643721 1 A n p fou lis 7 42 C m a a e s 4 l u U 1 1 p n u a mu EU 1 7 73 1 1 a a or a ta AM2 6 3 7 a a u r 3 3 3 ri b ca a 4 4 C m n la e s 725 e m a li i EU 3 0 1 am p u a 8 13 di fi ol 7 0 C a la la 713 7 ul f iata is EU 349430 1 n ct J5 U Fe la n la r s 71 8 a panu u lac i F E EU 64371 1 u a e n ii E 1 W m la flo U 4 p p n 7 34 p t r 9 an m s erfol A U 1 W a a p ifl a E 3 p a p oe m 71 3 26 1 Rh h n la ri o 3 m ni s ho u EU M 4 a le u pe mu ra 1 C i rad rc n 2 34 1 1 h n la 7 ampanuda e E 3 3 l b l Ca 1 io an lo a ta 71 R e e p reg if U C r o v ic U 49 22 1 igioph n e ol E 1 43 T c re r anuma a E hi b rg r i 6 2 1 iod e ic lc U7 7 34 45 S g e i re i a 1 r s a a E 1 1 ip rg a g n 7 i m cat U 3 2 S iop a 24 8 T ul a fa id E 134 4 1 S ho yllum ia h rin 7 5 63 n a al r a 7 1 iph h e 8 J 3 dan i f E U 1 4 c h d a 5 F amer s rid a 7 0 1 o y e e J o pa um ia yb E U 3 4 N iph o ll d r 13 ri m ou s h b rid is 1 4 6 1 codo d u a F FJ587253 T m y r A U 7 3 2 e o o m squarrose c U7 str ru g ia b e ia 1 4 1 H s n ra e 55160 1 Ca la t ou s h y A M 713 3 2 3 e o c s a E 6 1 h n n 4 Ber o s qu c Y as Le eg e o m EU7 M 2 4 1 te c d n p e 64 1 nu ou sia v g u 3 0 o o a a A a 1 u ia t m A 2 4 8 1 W r d de a Polynesia, southeast Asia and eastern Brazil (N4 in Figure 3 g o n r p 8 o s ta a u 3 4 0 e o t r 3 t E M2 9 9 1 1 W a ni c bi io ros u 1 366 m 1 Le g um n g a um a 1 4 h h n d m 3 panul 4 2 1 L ou l ir li t E U 4 C a c a ebi l id 7 a e g a a 3 9 6 le m is u U 3 1 g u pe v r A U 6 3 3 4 ra h e a e e m C 1 15 L e c i fo n at s 4 T l n n a l s E am 6 a v EU7 Y6 7 4 9 0 1 e u i U71 1 0 1 L e ia ni in a 9 he ter b r i s C 55 p E 1 3 9 1 T n e gu a ri E 3 1 s m o p de 7 4 b t EU7 6 s u u tic A U7 5 3 7 1 M r i o r e i 29 37 ma pinn oi 4 0 e le e g t n a 7 Y 4 16 4 a o la li e um E 5 c r i a n i r t M 1 1 7 1 M i ic r ap g a s 3 A 6 6 g ne lim u a e um A 3 1 c a i u 5 43 s y eu l c a sa i U 2 1 2 1 1 i h i b f 4 419 1 U u e n a b r A 40 6 Wa c ro a o s 6 3 3 L ar u ic at Y 7 3 3 1 e g s e E o m r o a A W r l l li As l l p c le m M 65 1 49 4 o c ia u a i r a Q g 1 sy u m a mo a E 7 o n t 713 71 u s u u Y 3 0 1 W h co th tar e EU D e 1 e o a pi r u a A 2 1 a d ifol U 7 r m n o U 5 4 6 le l r r U L t s o g s a E 6 3 44 W h on o o 6 9 o m a co c l i l Y 1 a d ie E E r f u s A 7 5 1 0 le n ng s i i 1 3 1 A yn e u p a s s i x U 4 W a h on o a 3 s i ff el e E 6 1 5 53 2 b i P r i M 9 1 P l nb de 3 4 et e a a E 7 h e e g e 5 46 A m m x a e de i U 5 3 1 3 a 1 3 ia l i E 1 r le n l b 6 1 P a euma u e r a d ch U 2 134 5 4 5 R h r g o 7 1 yn t l ul l lia E 9 1 1 i e g s 3 o U 71 s le b lo me ra 1 y e f a E 34942 1 0 7 M o n r 3 s C t n n u is noi ns i o 7 m e i 3 551 4 i i a EU U 5 1 M b g a m c 3 h op a o s eni E U 6 1 e n 1 5 1 A n c ul 1 3 er r i EU 6 1 i s AY a er t 4 s p v ra a 713 4 05 8 M ll b g a c ra e 7 EU7 P hy d a E 3 P e e 70 5 1 s pul ob s icif a u t a U 7 4 1 c a t l ap 3 7 y o D 3 e i ra at o p par r r f a E 7 4 1 r o g a t U 3 1 mp s te lo a c U 1 0 e i o AY 1 h si i t E 7 1 R P c rg u 3 c h r n i 13 7 r e a car ia E 4 51 a e s oc if c ta a D Q 6 338 0 4 r be e t a 7 m t a E U 13 Ro is i a m 43 o i t i y 6 3 e r u 5 3 P la p r o i a E U 1 r c mp 6 r r at i L 55 3 1 a ia g n n 3 a op a a i at i F Q 3 5 i m i m U 61 u pe a c li E U 6 4 1 s ra 6 C s AY6 U 3 1 er li si a EU7 1 6 1 P p g U te ig ca i a i FJ 1 a s c F 5 l G F J58 37 E 3 a

EU713441 1 Campanula lusitanica 4 F E m t E 12 1 P te n i o i l i 3 4 3 oe e g o o l t in r U th rica arr i 7 9 a u ul a 15 C a p mo v f t i l 8 4 7 P e l us s E 7 1 i s 0 1 4 33 1 r l 6 a a a e i J Pr 2 1 s 8 id e l a J r t l i 37 7 l r a s J u p U 5 AY a ii e li U 1 0 1 J l a t n o s 1 l e o ca 713 7 3 ism i 6 1 5 1 r o b x 6 a p m o c 5 1 8 a 5 u r i n o f n G p n re a 5 3 1 Fe l t e z 6 anul i 7 o ri f i u an d a va r i 3 1 l t n la ag a 7 9 71 i 52 f iv 6 s c i f H i 8 t 5 7 1 f 8 2 a o 437 a v 7271 1 C s fo 8 c EU a e bs a 1 s p a r r 8 1 1 i re r o y h i 11 c if p a l e 4 1 u i s a m a d 1 3 J m 33 1 4 u a d 7 16 a 5 2 0 a c a 6 7 an a 1 EU r i 7 u b o a 3 6 1 s p r a f c 7 e i l a u d J m at ci i 34 nu le h s 3 J a l rp l r t i 3 7 a fo div 3 4 vi u n 2 7 4 m p 1 1 si i a EU7 p s o 2 a li i a 4 2 4 1 3) all derive from a single ancestor (Bayesian posterior o s a u 2 7 a r 1 n n a cos h r n 5 J s und m li u o 71 am m la d m rpus a l 4 1 li 3 7 36 ocar a a u 6 7 a i i 4 a fo 1 20 1 h l a 5 t u l 6 3 1 J s o EU Ca u e 7 1 a t 7 a a c l t u as i at i s pa a o a n u a 1 4 T a 1 on k s a 4 t t 6 7 J to s p u ot 5 1 a i n ph 3 C b r 8 1 h 2 l C am p o 1 U u c on c C a n cu nu m C a i r 4 a i a 4 r 3 c o u EU m o r s e a nula n y o i o a c w i d U71341 1 C co 1 a T c ar n r 1 1 a p 1 Tr E 1 a e o n a 1 e 1 s 1 a r m T i s n a a a s l n pu n p n a a if a 1 t 1 o l di E c r e a a m m

p d i n g 15 71 7134 8 1 pa a C p EU7 e a l C Ca r m fu 2 a a o m C e 1 C pa u e p r C s h l a l C ac C C ampa h ne p l u 2 4 a a e a u ul a f 1 p C u U u C pa c U 8 d a l c n o s d m l n C a am f u m a e s a H u n p a e s m l a c h u a a u a r s n h n E m u e u a h 1 l E A a h a u r A C n a mp m l is s m 13 64 n a h a e vi t 37 m s 43 a u m s 1 m i m 1 a e nu t if 8 a n m e s n u m u s p e o a m a c u 4 7 2 a p e n l

3 n 1 pan pa p Ca p d s o 872 Cam C p l s c 6 C p a h p l 1 a p m l n h 9 p p o 6 7 1 C i ula n s 1 2 p l iu d a U m a p u p li 1 a an n i la r e h m a o l 0 p a a p p a 5 7 4 m a u n a e 7 8 3 m re e a

p a a n m J5 m a s l E 1 n ul 1 n m n 72 3 m a o n n c e 1 4 p 5 4 2 2 n m t ic c 8 1 m s U m n ul a EU a n f F 0 1 u u t 8 4 a u a i ch n u u 7 3 m a ht J u C a rut 3 3 7 p a c i Ca C a m u n h u E 5 51 Ca l l c l e 3 C l e l l a 1 a i F 37 l a a h 4 C r c u 1 a n a a s e a s i l a a 4 4 Ca 1 l a te d 1 1 1 C r i 43 a 7 a a 4 b i 37 4 1 7 a i a C r 1 a u p i e cu FJ5 1 p e 3 3 g 7 6 2 C o el i

r e A 6 4 3 i r 1 0 p 1 1342 2 i e U s l c l o l 1 3 a r i 1 a 7 6 1 l z e U 6 1 u a r U 3 1 unc U y a u 1 EU 7 4 1 p r a l s E 4 p u 7 5 r oi 9 os 4 2 u s t u 7 7 4 E m t l E 0 g U a lyx E c i l 4 u e U 7 i 2 i m 5 U7 U 7 5 n e l de 2 3 n t 5 t a o E U h n u 3 p a 2 a 72 4 4 E 8 4 u e u E ul E 7 1 e 134 2 n a i

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i s Ancestral state reconstructions indicate that this ancestor MaximumFigure 2 Likelihood trees of the Campanulaceae was most likely confined to Africa and that it was a nano- Maximum Likelihood trees of the Campanulaceae. phanerophyte (in 950 and 816 of 1000 reconstructions, Cladograms with the highest likelihood scores yielded from respectively; the other reconstructions being ambiguous 10 independent runs in the software GARLI, based on: (a) rather than a different state; see Figures 7 and 9). Indeed, trnL-F, 452 sequences; (b) ITS, 445 sequences; and (c) rbcL, it is in this clade that the truly giant habit occurs, as exhib- 438 sequences. Subfamily Lobelioideae is highlighted in green. ited by L. gloria-montis and L. rhynchopetalum (Figure 1). GenBank accession numbers are given following the species This corroborates previous results, based on fewer species, names (as stored in GenBank). that giant lobelias are closely related and are ultimately derived from herbaceous ancestors [7,18,21] but contra-

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Helianthus annuus 0.94 Canarina canariensis 1.00 Jasione montana – 0.97 Legousia hybrida 100 0.99 Campanula asperuloides CAMPANULOIDEAE 67 1.00 Campanula latifolia 80 100 Campanula trachelium 1.00 Lobelia physaloides 1.00 Lobelia coronopifolia 95 100 Lobelia tomentosa 1.00 1.00 Grammatotheca bergiana N1 Lobelia aquatica 97 100 Lobelia sonderiana 1.00 1.00 Lobelia erinus Lobelia graniticola 100 100 Lobelia anceps 1.00 Lobelia columnaris 1.00 Lobelia gibberoa 96 66 Lobelia gregoriana 0.89 Lobelia boninensis Lobelia petiolata 0.67 58 Lobelia leschenaultiana 1.00 60 Lobelia nicotianifolia 1.00 1.00 Lobelia gloriamontis 92 Lobelia kauaensis 98 N4 100 Lobelia villosa Apetahia longistigmata 1.00 Apetahia margaretae 98 1.00 Sclerotheca forsteri 87 Sclerotheca jayorum 0.81 1.00 Lobelia stricklandiae 1.00 N5 1.00 Lobelia exaltata N2 – 82 0.99 Lobelia fistulosa 90 100 Lobelia organensis 871.00 Brighamia insignis 1.00 98 Brighamia rockii 1.00 Delissea rhytidosperma 1.00 Delissea subcordata 88 Delissea undulata 1.00 Trematolobelia kauaiensis 0.50 99 Trematolobelia macrostachys 1.00 Lobelia niihauensis – 1.00 Lobelia hypoleuca 97 88 Lobelia yuccoides 1.00 Cyanea angustifolia 1.00 Cyanea coriacea 98 Cyanea kuhihewa 1.00 91 Cyanea leptostegia 1.00 N6 1.00 Clermontia fauriei N3 75 1.00 Clermontia arborescens 1.00 0.92 100 1.00 Clermontia kakeana 100 Clermontia parviflora 73 97 Cyanea hirtella 1.00 Cyanea acuminata 100 0.85 Cyanea koolauensis 72 0.98 Cyanea floribunda Cyanea pilosa 64 Diastatea micrantha LOBELIOIDEAE 0.57 Solenopsis laurentia 1.00 Downingia bacigalupii 1.00 82 N8 100 Downingia insignis 63 1.00 Lobelia kalmii 1.00 Lobelia rotundifolia 100 1.00 Lobelia cardinalis 1.00 100 Lobelia dortmanna 100 Lobelia cordifolia 82 Lobelia xalapensis 0.99 0.99 Lobelia irasuensis 1.00 Lobelia aguana 56 93 Lobelia laxiflora 97 Hippobroma longiflora Lobelia kraussi 1.00 Lobelia martagon 0.97 99 Lobelia portoricensis N7 0.73 Lobelia stricta – – Lobelia vivaldii 1.00 Lobelia excelsa N10 0.71 Lobelia polyphylla 0.66 100 Lobelia tupa 76 Lobelia oligophylla – 1.00 Lobelia nummularia N11 0.82 100 0.97 Lobelia macrodon 58 Lobelia roughii 54 Isotoma axillaris 1.00 Isotoma fluviatilis Lobelia purpurascens 66 0.82 Lobelia chinensis 1.00 N9 0.98 Lobelia perpusilla 67 – 1.00 Lobelia angulata – Lobelia arenaria 81 Centropogon dissectus Centropogon luteus Centropogon trichodes Siphocampylus affinis Siphocampylus brevicalyx 1.00 N12 Siphocampylus fulgens Siphocampylus giganteus 99 1.00 Lysipomia cuspidata Lysipomia sphagnophila 0.92 100 Siphocampylus macropodus 58 Siphocampylus scandens 1.00 Centropogon cornutus 1.00 Centropogon gamosepalus 56 91 Centropogon granulosus 0.86 Centropogon gutierrezii 1.00 Burmeistera domingensis – 1.00 Burmeistera crispiloba 97 86 Burmeistera cyclostigmata PhylogenyFigure 3 of subfamily Lobelioideae Phylogeny of subfamily Lobelioideae. Fifty-percent majority-rule consensus cladogram from the Bayesian analysis based on the combined data set (trnL-F, ITS, rbcL). Numbers above branches indicate Bayesian posterior probabilities; numbers below branches represent bootstrap support values (> 50) calculated under maximum parsimony. Key nodes discussed in the text are labelled N1--N12.

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Helianthus annuus Canarina canariensis Jasione montana C1 C2 Legousia hybrida Campanula asperuloides Campanula latifolia Campanula trachelium Lobelia physaloides Lobelia coronopifolia Lobelia tomentosa Grammatotheca bergiana Lobelia aquatica Lobelia sonderiana Lobelia erinus Lobelia graniticola Lobelia anceps Lobelia columnaris Lobelia gibberoa Lobelia gregoriana Lobelia boninensis Lobelia petiolata Lobelia leschenaultiana Lobelia nicotianifolia C3 Lobelia gloriamontis Lobelia kauaensis Lobelia villosa Apetahia longistigmata Apetahia margaretae Sclerotheca forsteri Sclerotheca jayorum Lobelia stricklandiae Lobelia exaltata Lobelia fistulosa Lobelia organensis Brighamia insignis C4 Brighamia rockii Delissea rhytidosperma Delissea subcordata Delissea undulata C5 Trematolobelia kauaiensis Trematolobelia macrostachys C6 Lobelia niihauensis Lobelia hypoleuca Lobelia yuccoides Cyanea angustifolia Cyanea coriacea Cyanea kuhihewa C7 Cyanea leptostegia Clermontia fauriei Clermontia arborescens Clermontia kakeana Clermontia parviflora Cyanea hirtella Cyanea acuminata Cyanea koolauensis Cyanea floribunda Cyanea pilosa Diastatea micrantha Solenopsis laurentia Downingia bacigalupii Downingia insignis Lobelia kalmii Lobelia rotundifolia Lobelia cardinalis Lobelia dortmanna Lobelia cordifolia Lobelia xalapensis Lobelia irasuensis Lobelia aguana Lobelia laxiflora Hippobroma longiflora Lobelia kraussi Lobelia martagon Lobelia portoricensis Lobelia stricta Lobelia vivaldii Lobelia excelsa Lobelia polyphylla Lobelia tupa Lobelia oligophylla Lobelia nummularia Lobelia macrodon Lobelia roughii Isotoma axillaris Isotoma fluviatilis Lobelia purpurascens Lobelia chinensis Lobelia perpusilla Lobelia angulata Lobelia arenaria Centropogon dissectus Centropogon luteus Centropogon trichodes Siphocampylus affinis Siphocampylus brevicalyx Siphocampylus fulgens Siphocampylus giganteus Lysipomia cuspidata Lysipomia sphagnophila Siphocampylus macropodus Siphocampylus scandens Centropogon cornutus Centropogon gamosepalus Centropogon granulosus Centropogon gutierrezii Burmeistera domingensis Burmeistera crispiloba Burmeistera cyclostigmata

TIME (Ma) 100 90 80 70 60 50 40 30 20 10 0

DivergenceFigure 4 time estimates using Penalized Likelihood Divergence time estimates using Penalized Likelihood. Mean age chronogram showing 95% confidence intervals of age nodes (bars), based on 1000 Bayesian trees from a post burn-in tree sample. The stars represent calibration points: C1, crown age of Asterales as estimated by Bremer et al. [66], fixed age = 93 Ma; C2, crown age of subfamily Campanuloideae, based on a fossil Campanula, minimal age = 5.33 Ma [68]; C3 -- C7, diversification of Hawaiian taxa, corresponding to the age of the oldest island of the Hawaiian Ridge (Kure) after which a continuous chain of islands has been available as 'stepping stones' for prop- agules of the Hawaiian biota, maximum age = 29.8 Ma [26].

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Helianthus annuus Canarina canariensis C1 Jasione montana C2 Legousia hybrida Campanula asperuloides Campanula latifolia Campanula trachelium Lobelia physaloides Lobelia tomentosa Lobelia coronopifolia Lobelia aquatica Grammatotheca bergiana Lobelia sonderiana Lobelia graniticola Lobelia erinus Lobelia anceps Lobelia columnaris Lobelia gregoriana Lobelia gibberoa Lobelia stricklandiae Lobelia exaltata Lobelia organensis Lobelia fistulosa Lobelia nicotianifolia Lobelia leschenaultiana Apetahia longistigmata Apetahia margaretae Sclerotheca forsteri Sclerotheca jayorum Lobelia petiolata Lobelia boninensis Brighamia rockii Brighamia insignis Delissea undulata Delissea rhytidosperma Delissea subcordata Lobelia niihauensis C3 Lobelia hypoleuca Lobelia yuccoides Trematolobelia macrostachys Trematolobelia kauaiensis Lobelia villosa Lobelia gloriamontis Lobelia kauaensis Cyanea angustifolia Cyanea kuhihewa Cyanea coriacea Cyanea leptostegia Clermontia fauriei Clermontia arborescens Clermontia parviflora Clermontia kakeana Cyanea hirtella Cyanea pilosa Cyanea floribunda Cyanea koolauensis Cyanea acuminata Diastatea micrantha Solenopsis laurentia Downingia bacigalupii Downingia insignis Lobelia kalmii Lobelia rotundifolia Lobelia cardinalis Lobelia dortmanna Lobelia xalapensis Lobelia cordifolia Lobelia irasuensis Lobelia laxiflora Lobelia aguana Lobelia portoricensis Lobelia kraussi Hippobroma longiflora Lobelia martagon Lobelia stricta Lobelia vivaldii Lobelia polyphylla Lobelia excelsa Lobelia tupa Lobelia oligophylla Lobelia nummularia Lobelia roughii Lobelia macrodon Isotoma fluviatilis Isotoma axillaris Lobelia purpurascens Lobelia chinensis Lobelia perpusilla Lobelia angulata Lobelia arenaria Lysipomia cuspidata Lysipomia sphagnophila Siphocampylus fulgens Siphocampylus brevicalyx Siphocampylus affinis Siphocampylus giganteus Centropogon cornutus Centropogon granulosus Centropogon gamosepalus Centropogon gutierrezii Burmeistera domingensis Burmeistera cyclostigmata Burmeistera crispiloba Siphocampylus scandens Siphocampylus macropodus Centropogon trichodes Centropogon dissectus Centropogon luteus

TIME (Ma) 100 90 80 70 60 50 40 30 20 10 0 DivergenceFigure 5 time estimates using Bayesian relaxed clock (BEAST) Divergence time estimates using Bayesian relaxed clock (BEAST). Tree, with the maximum sum of clade credibilities and branch lengths equal to the median ages as calculated from 40,000 post burn-in chronograms. Bars show 95% Highest Pos- terior Density intervals of age nodes. Calibration points as in the previous figure, with the following exceptions: (i) that the tree prior incorporated for the root of the tree (C1, 93 Ma) was not constrained a priori on a particular clade, but allowed to be calculated in the phylogenetic and dating estimation; and (ii) that all Hawaiian species were constrained as monophyletic prior to the analysis, following the results by Givnish et al. [10] (see Methods).

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A

-0.2 -0.1 0 0.1 0.2 0.3 0.4 B

5 10 15 20 25 30 35 Age (Ma)

SelectedFigure 6 statistics from the BEAST analysis Selected statistics from the BEAST analysis. Distribution curves obtained from a post burn-in sample of 40,000 chrono- grams; each curve represents an independent run. (a) Covariance between parent and child branch rates. A value close to zero indicates that there is no support for autocorrelation (a main assumption in Penalized Likelihood analyses), meaning that the BEAST results here are probably more realistic. (b) Distribution of age estimates for the crown group of the Hawaiian taxa.

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Table 1: Crown group ages (in million of years) of the major reconstructed to Africa with confidence. Range shifts are groups outlined in Figure 3. unambiguously inferred from: Africa to Oceania (Lobelia Penalized Likelihood BEAST physaloides); Africa to the Neotropics (Lobelia aquatica); the Neotropics to Oceania (Lobelia macrodon - L. arenaria); Clade Mean 95% CI Median 95% HPD and Oceania to southeast Asia (Lobelia chinensis). That the Brazilian giant lobelias are together sister to an African N1 54.9 50.9 -- 59.6 72.7 52.2 -- 88.2 species is strongly supported, as previously suggested, by Knox et al. [18]. N2 43.9 39.0 -- 47.8 45.5 30.9 -- 59.2 There is no support here for the hypothesis that the giant N3 40.7 36.3 -- 44.8 39.7 27.4 -- 53.1 lobelias arrived in eastern Africa from Asia or the Pacific region [7]. However, the resolution at the base of clade N4 N4 32.7 27.1 -- 38.0 24.5 15.1 -- 36.6 is very poor and the relationships among the strongly sup- ported clades within it - and possibly their biogeographic N5 30.3 24.8 -- 35.9 20.8 12.4 -- 30.5 reconstructions - may substantially change with the addi- tion of more species and sequence data. Indeed, in the N6 22.4 16.9 -- 29.0 12.2 5.74 -- 16.2 analysis by Givnish et al. [10], L. nicotianifolia (a southeast Asian species) was inferred to be sister to a clade compris- N7 39.6 34.9 -- 43.6 36.7 25.1 -- 49.5 ing French Polynesian, African and Hawaiian subclades. N8 37.4 32.8 -- 41.8 32.8 22.0 -- 45.4 In the present analysis, that 'basal' position is occupied by African species (L. columnaris, L. gibberoa and L. gregori- N9 39.0 31.0 -- 40.4 29.6 18.9 -- 41.3 ana), although the support for this placement is weak (Bpp = 0.81, Bs < 50). As taxon sampling, sequence N10 11.8 3.48 -- 20.6 4.56 0.95 -- 10.9 regions and phylogenetic methods have all varied consid- erably between different studies, further investigation is N11 19.0 12.7 -- 25.1 15.5 8.43 -- 23.8 clearly needed in order to reconstruct a solid biogeo- graphic scenario for the large lobelioids. N12 27.4 22.5 -- 32.0 18.8 10.6 -- 29.0 Dispersal versus vicariance BEAST = Bayesian evolutionary analysis by sampling trees; CI = Although the exact timing for transcontinental range confidence interval; HPD = highest posterior density. shifts inferred here are prone to large error margins asso- dicts some earlier suggestions the giant habit would be ciated with topological and branch length uncertainties plesiomorphic in the family [6]. (see the credibility bars at nodes in Figures 4 and 5), all range shifts are estimated to have occurred in the last 50 Nanophanerophyte ancestors (with buds below 3 m high; Ma and, in several cases, presumably much more recently. see Table 2) have given rise to phanerophytes (buds above Given that the final break-up of Gondwanaland took 3 m) several times independently. Although several spe- place around ~100 Ma [24], these results provide evidence cies are strictly phanerophytic (for example, L. giberroa, L. that long-distance dispersals must have played a major petiolata, L. stricklandiae, Delissea undulata and Cyanea lep- role in shaping the present-day distribution of taxa, a sce- tostegia), others often vary in height (for example, Scle- nario also corroborated by the molecular dating analysis rotheca jayorum, Brighamia insignis, Trematolobelia of Givnish et al. [10]. This contrasts to early suggestions macrostachys, Cyanea angustifolia and Siphocampylus gigan- [6] that distribution patterns may be the result of vicari- teus). These results indicate that, in the Lobelioideae, ance caused by continental drift. height seems to be a more labile state than woodiness. Long-distance dispersal seems plausible for lobelioids, Biogeographic history since most species produce large amounts of minuscule Geographic range development of the Lobelioideae is seeds. From herbarium specimens, I calculated that 1 g of depicted in Figure 9. As with life form, distribution is phy- seed of the Hawaiian endemic Clermontia kakeana con- logenetically conservative (P < 0.001; Figure 8c-d), but tains about 36,000 seeds (Figure 10). Although seeds of even more so given its higher consistency index (0.40) Clermontia and some other Hawaiian genera (Cyanea and and fewer parsimony steps required for reconstructing the Delissea) are today contained in bird-dispersed berries, Bayesian cladogram (25). other Hawaiian taxa (Lobelia, Trematolobelia and Brigha- mia) still possess wind-dispersed capsules, a condition The MRCA of the Lobelioideae, as well as those of most of also inferred for the MRCA of the Hawaiian clade by the early diverging splits (N1 - N3 in Figure 3), are all Givnish et al. [10]. It is therefore reasonable to conclude

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Lobelia physaloides Lobelia coronopifolia Lobelia tomentosa Grammatotheca bergiana Lobelia aquatica Lobelia sonderiana Lobelia erinus Lobelia graniticola Lobelia anceps Lobelia columnaris Lobelia gibberoa Lobelia gregoriana Lobelia boninensis Lobelia petiolata Lobelia leschenaultiana Lobelia nicotianifolia Lobelia gloriamontis Lobelia kauaensis Lobelia villosa Apetahia longistigmata Apetahia margaretae Sclerotheca forsteri Sclerotheca jayorum Lobelia stricklandiae Lobelia exaltata Lobelia fistulosa Lobelia organensis Brighamia insignis Brighamia rockii Delissea rhytidosperma Delissea subcordata Delissea undulata Trematolobelia kauaiensis Trematolobelia macrostachys Lobelia niihauensis Lobelia hypoleuca Lobelia yuccoides Cyanea angustifolia Cyanea coriacea Cyanea kuhihewa Cyanea leptostegia Clermontia fauriei Clermontia arborescens Clermontia kakeana Clermontia parviflora Cyanea hirtella Cyanea acuminata Cyanea koolauensis Cyanea floribunda Cyanea pilosa Diastatea micrantha Solenopsis laurentia Downingia bacigalupii Downingia insignis Lobelia kalmii Lobelia rotundifolia Lobelia cardinalis Lobelia dortmanna Lobelia cordifolia Lobelia xalapensis Lobelia irasuensis Lobelia aguana Lobelia laxiflora Hippobroma longiflora Lobelia kraussi Lobelia martagon Lobelia portoricensis Lobelia stricta Lobelia vivaldii Lobelia excelsa Lobelia polyphylla Lobelia tupa Lobelia oligophylla Lobelia nummularia Lobelia macrodon Lobelia roughii Isotoma axillaris Isotoma fluviatilis Aquatic Lobelia purpurascens Lobelia chinensis Biennal Lobelia perpusilla Chamaephyte Lobelia angulata Lobelia arenaria Geophyte Centropogon dissectus Hemicryptophyte Centropogon luteus Centropogon trichodes Herbaceous Phanerophyte Siphocampylus affinis Siphocampylus brevicalyx Nanophanerophyte Siphocampylus fulgens Phanerophyte Siphocampylus giganteus Lysipomia cuspidata Therophyte Lysipomia sphagnophila Siphocampylus macropodus Node Absent Siphocampylus scandens Centropogon cornutus Equivocal Centropogon gamosepalus Centropogon granulosus Centropogon gutierrezii Burmeistera domingensis Burmeistera crispiloba Burmeistera cyclostigmata

50.0 40.0 30.0 20.0 10.0 0.0 LifeFigure form 7 shifts in relation to time Life form shifts in relation to time. Results from character state reconstructions in subfamily Lobelioideae. The pie charts on each node show the relative proportion of character assignments based on the individual results from the Fitch optimiza- tion of 1000 Bayesian post burn-in trees, counting uniquely best states. States of extant species are shown before each species name. Results plotted on the mean age chronogram obtained using Penalized Likelihood (Figure 4). See Table 2 for a definition of life forms.

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250 240 230 AB 220 500 210 200 190 180 400 170 160 150 140 130 300 120 110 100

90 200 80 Number of Trees 70 60 50 100 40 30 20 10 82 83 84 85 86 87 88 89 90 91 92 93 94 95 66 67 68 69

190 180 CD 170

160 500

150

140

130 400

120

110

100 300 90

80

70

Number of Trees 200 60

50

40

30 100

20

10 58 59 60 61 65 66 67 68 70 71 62 63 64 69 23 24 25 26 27 Number of Steps Number of Steps

TestsFigure of 8phylogenetic conservatism in life form and geographic distribution Tests of phylogenetic conservatism in life form and geographic distribution. Comparison of the minimal number of steps required to reconstruct life form and geographic distribution on a sample of 1000 simulated trees (generated by keeping the tree topology as in the Bayesian consensus, but randomly shuffling character states) with 1000 empirical trees (randomly chosen from the post burn-in Bayesian sample). (a) Simulated results for life form; (b) empirical results for life form; (c) simu- lated results for geographic distribution; and (d) empirical results for geographic distribution. For both characters, the observed values fall outside the lower percentiles of the simulated curves (indicated by the red lines in (a) and (c); P < 0.001), indicating that both life form and geographic distribution are phylogenetically conservative. that wind dispersal of tiny seeds was the dispersal mode BEAST analysis, where the Hawaiian taxa had to be con- for the ancestor of the Hawaiian lobelioids, as suggested strained as monophyletic (see Methods), the crown group by Givnish et al. [10] and that, presumably, the same of the whole Hawaiian clade had a mean of 16.4 Ma (9.43 applies for other lobelioid dispersal events. Millions of - 23.0 Ma, 95% HPD; see Figure 6b). These ages are only tons of dust are transported annually from the Sahara into slightly older than the estimates by Givnish et al. [10], South America [25], so it is conceivable that, given which varied between 13.6 ± 3.11 Ma and 13.0 ± 1.00 enough time, lobelioid seeds could also be carried over depending on calibration methodology. the 15,000 km separating the Hawaiian Islands and Africa and, subsequently, succeed in germinating and establish- The relatively old node ages estimated here may seem sur- ing a new population. prising, given that the oldest of the modern Hawaiian Islands with well-developed vegetation only dates back to Colonization of the Hawaiian Islands 5.1 Ma [26]. However, they are fully explainable when The earliest diversification of lineages endemic to the considering that these islands are part of a much older Hawaiian Islands corresponds to the crown age of node archipelago formed by the movement of the Pacific plate N6 retrieved in the MrBayes analysis (Figure 3). In the PL over a fixed hot spot. A continuous chain of islands have analysis, that clade had a mean of 22.4 Ma (16.9 - 29.0 been elevated since 29.8 Ma [26] and lobelioid lineages Ma, 95% confidence intervals, CI), whereas in the BEAST could have continuously colonized the rising islands and analysis the mean was 12.2 Ma (5.74 - 16.2 Ma, 95% ceased to exist in the subsiding ones. Island hopping by a Highest Posterior Density, HPD; Table 1). However, in the plant adapted to long-distance dispersal is biologically

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Table 2: Characterization of life forms used in the ancestral state reconstructions (from Lammers [23]).

Life form Stems Buds

Aquatic Comprises both Hydrophytes and Hydrohemicryptophytes

Chamaephyte Herbaceous and/or woody and persistent On or just above soil level but never more than 0.5 m above ground

Geophyte Hemicryptophytes that survive unfavourable seasons in the form of Below soil level a rhizome, bulb, tuber or root bud

Hemicryptophyte Herbaceous, often dying back after the growing season but with Just on or below soil level buds or growth at soil level

Herbaceous phanerophyte Herbaceous and persisting for several years Above soil level

Hydrophyte Vegetative shoots entirely in water, the leaves usually submersed Permanently or temporarily on the bottom of and/or floating; flower-bearing parts may emerge above the water the water

Nanophanerophyte Woody and indefinitely persistent Above soil level but normally less than 3 m above ground

Phanerophyte Woody and indefinitely persistent Normally 3 m or more above ground

Therophyte Plants surviving unfavourable seasons in the form of seeds, completing their life-cycle during the favourable season feasible and has been suggested for the Hawaiian genus tive role of competing hypotheses of drivers of diversifica- Hillebrandia in the plant family Begoniaceae [27] and the tion in this clade, be they mainly abiotic (for example, fern genus Diellia [28]. geotectonic events, climatic fluctuations) or biotic (for example, pollinator interactions, such as those demon- Correlates of lobelioid diversification strated for certain species of Centropogon and Burmeistera In order to identify possible drivers of biotic diversifica- [36-40]). tion, it is essential to infer when and where lineages diver- sified. Ages and ancestral areas reconstructed here may, The temporal framework for the Lobelioideae provided therefore, provide a suitable starting point for further here may also contribute to the discussion on the diversi- examination of particular clades in the Lobelioideae. fication of particular Lobelioideae groups, for which a fine-scale biogeographic analysis has been performed but One appealing example of how these reconstructions can for which no absolute divergence times have been esti- be useful concerns the SCBL clade (Siphocampylus, Centro- mated. One of these is the giant lobelias of eastern Africa, pogon, Burmeistera and Lysipomia - clade N12 in Figure 3). which have been thoroughly studied by Knox and co- This group of mainly nanophanerophytes and chamae- workers [7,13,18,41]. Crown ages for the eastern African phytes comprises over 580 species, which is about half of clade, comprising Lobelia columnaris, L. gibberoa, and L. all Lobelioideae species [21,23,29-31]. The SCBL clade is gregoriana, estimated here (in PL: mean 22.3 Ma; 13.7 - entirely confined to the Neotropical region, and is partic- 30.4 Ma, 95% CI; in BEAST: median 11.2 Ma; 3.09 - 20.8 ularly rich in species in the Northern Andes. The uplift of Ma, 95% HPD) clearly predate the age of most of the the Northern Andes began at ~31 Ma and intensified in mountains in the Eastern and Western African Rifts [7]. the last ~20 Ma, with several discrete phases of uplift These findings influence the interpretation of the tempo which especially affected the Eastern Cordilleras [32]. The and possible processes underlying diversification in the temporal match between the Andean uplift and the ages region (see [7] for a detailed account on competing inferred here is striking: onset of diversification was hypotheses and their predictions). inferred at ~27 Ma (PL) or ~19 Ma (BEAST) (Table 1) and the MRCA of this clade was unambiguously inferred as the Conclusion Neotropics (Figure 9). Although a correlation in time and Let us recapitulate the thought-provoking hypothesis that space does not necessarily imply causation, there is an the giant lobelias of Africa and the Hawaiian Islands con- emerging consensus that the Andean uplift has played a verged into the giant life form from herbaceous ancestors major role in Neotropical diversification [32-35]. Further in the same way as the giant senecios and the silversword studies are clearly needed in order to disentangle the rela- alliance have (pages 84-85 in [4]; Figure 1). The distant

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Table 3: List of species used in the analyses of the Lobelioideae data set, their life form and distribution (simplified from Lammers [23]), and GenBank accession numbers.

Taxon Life form Distribution rbcL trnL-trnF ITS

1 Apetahia longistigmata Nanophanerophyte French Polynesia DQ285272 DQ285155 2 Apetahia margaretae Nanophanerophyte French Polynesia DQ285286 DQ285169 3 Brighamia insignis Nanophanerophyte or Hawaiian islands AF042664 DQ356189 EU219385* Phanerophyte 4 Brighamia rockii Nanophanerophyte or Hawaiian islands DQ285257 DQ285140 Phanerophyte 5 Burmeistera crispiloba Nanophanerophyte Neotropics EF174641 DQ285164 6 Burmeistera cyclostigmata Nanophanerophyte or Neotropics DQ356147 DQ356213 Chamaephyte 7 Burmeistera domingensis Nanophanerophyte or Neotropics DQ356148 DQ356214 Chamaephyte 8 Campanula asperuloides Hemicryptophyte Temp. Eurasia DQ356117 DQ356170 9 Campanula latifolia Hemicryptophyte Temp. Eurasia EF141027 DQ356169 10 Campanula trachelium Hemicryptophyte Temp. Eurasia DQ356118 DQ356171 11 Canarina canariensis Geophyte Temp. Eurasia DQ356115 DQ356167 12 Centropogon cornutus Nanophanerophyte or Neotropics DQ356158 DQ356226 Chamaephyte 13 Centropogon dissectus Nanophanerophyte or Neotropics EF141026 DQ356215 Chamaephyte 14 Centropogon gamosepalus Nanophanerophyte or Neotropics DQ356157 DQ356225 Chamaephyte 15 Centropogon granulosus Nanophanerophyte Neotropics DQ356152 DQ356220 16 Centropogon gutierrezii Chamaephyte Neotropics AF042658 DQ285165 17 Centropogon luteus Nanophanerophyte Neotropics DQ356151 DQ356219 18 Centropogon trichodes Nanophanerophyte or Neotropics DQ356149 DQ356217 Chamaephyte 19 Clermontia arborescens Nanophanerophyte or Hawaiian islands DQ285258 DQ285141 Phanerophyte 20 Clermontia fauriei Nanophanerophyte or Hawaiian islands DQ285259 DQ285142 Phanerophyte 21 Clermontia kakeana Nanophanerophyte or Hawaiian islands L18789 DQ356172 EU219386* Phanerophyte 22 Clermontia parviflora Nanophanerophyte Hawaiian islands DQ285288 DQ285171 23 Cyanea acuminata Nanophanerophyte Hawaiian islands DQ285261 DQ285144 24 Cyanea angustifolia Nanophanerophyte or Hawaiian islands DQ356119 DQ356173 EU219384* Phanerophyte 25 Cyanea coriacea Nanophanerophyte or Hawaiian islands AF042662 DQ285145 Phanerophyte 26 Cyanea floribunda Nanophanerophyte Hawaiian islands DQ285290 DQ285173 27 Cyanea hirtella Nanophanerophyte or Hawaiian islands DQ285292 DQ285175 Phanerophyte 28 Cyanea koolauensis Nanophanerophyte Hawaiian islands DQ356128 DQ356193 29 Cyanea kuhihewa Nanophanerophyte Hawaiian islands DQ285294 DQ285177 30 Cyanea leptostegia Phanerophyte Hawaiian islands DQ285289 DQ285172 31 Cyanea pilosa Nanophanerophyte Hawaiian islands DQ285291 DQ285174 32 Delissea rhytidosperma Nanophanerophyte Hawaiian islands AF042663 DQ285146 33 Delissea subcordata Nanophanerophyte Hawaiian islands DQ285264 DQ285147 34 Delissea undulata Phanerophyte Hawaiian islands DQ356188 EU219389* 35 Diastatea micranta Therophyte Neotropics DQ356138 DQ356203 36 Downingia bacigalupii Therophyte N America EF141031 DQ356183 37 Downingia insignis Therophyte N America EF141030 DQ356185 38 Grammatotheca bergiana Hemicryptophyte Africa DQ356116 DQ356168 AF163429 39 Helianthus annuus - (other) (other) AF097517 AJ430967 Outgroup 40 Hippobroma longiflora Therophyte, Biennal or Neotropics DQ356140 DQ356206 Hemicryptophyte 41 Isotoma axillaris Hemicryptophyte Oceania DQ268874 DQ285166 42 Isotoma fluviatilis Hemicryptophyte Oceania DQ356161 DQ356230 43 Jasione montana Biennal or Therophyte Temp. Eurasia DQ356120 DQ356174 44 Legousia hybrida Therophyte Temp. Eurasia DQ356163 DQ356234

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Table 3: List of species used in the analyses of the Lobelioideae data set, their life form and distribution (simplified from Lammers [23]), and GenBank accession numbers. (Continued)

45 Lobelia aguana Nanophanerophyte or Neotropics DQ356122 DQ356176 Chamaephyte 46 Lobelia anceps Hemicryptophyte DQ356124 DQ356184 47 Lobelia angulata Hemicryptophyte Oceania AY568754+AY568744 48 Lobelia aquatica Aquatic Neotropics EF141029 DQ356182 49 Lobelia arenaria Hemicryptophyte Oceania AY568756+AY568737 50 Lobelia boninensis Nanophanerophyte or Bonin Islands AF042661 DQ285157 Herbaceous phanerophyte 51 Lobelia cardinalis Hemicryptophyte N America & Neotropics AF042659 DQ356231 52 Lobelia chinensis Hemicryptophyte or Southeast Asia DQ356228 Geophyte 53 Lobelia columnaris Nanophanerophyte or Africa DQ285275 DQ285158 Herbaceous phanerophyte 54 Lobelia cordifolia Therophyte Neotropics DQ356204 55 Lobelia coronopifolia Chamaephyte Africa EF141025 DQ356181 56 Lobelia dortmanna Aquatic N America & Temp. DQ356162 DQ356232 EU219388* Eurasia 57 Lobelia erinus Hemicryptophyte or Africa L01931 DQ356233 Therophyte 58 Lobelia exaltata Herbaceous phanerophyte or Neotropics DQ356135 DQ356200 EU219383* Hemicryptophyte 59 Lobelia excelsa Nanophanerophyte or Neotropics DQ356146 DQ356212 Phanerophyte 60 Lobelia fistulosa Herbaceous phanerophyte or Neotropics DQ356136 DQ356201 EU219387* Chamaephyte 61 Lobelia giberroa Phanerophyte Africa DQ356127 DQ356192 EU219380* 62 Lobelia gloria-montis Nanophanerophyte Hawaiian islands DQ285265 DQ285148 63 Lobelia graniticola Hemicryptophyte Africa DQ356129 DQ356194 64 Lobelia gregoriana Nanophanerophyte or Africa DQ356187 EU219379* Herbaceous phanerophyte 65 Lobelia hypoleuca Nanophanerophyte Hawaiian islands DQ356126 DQ356191 66 Lobelia irasuensis Hemicryptophyte or Neotropics DQ356121 DQ356175 AY362765 Geophyte 67 Lobelia kalmii Hemicryptophyte N America DQ356166 EF126736 68 Lobelia kauaensis Nanophanerophyte Hawaiian islands DQ285267 DQ285150 69 Lobelia kraussii Hemicryptophyte Neotropics EF141024 DQ356179 70 Lobelia laxiflora Nanophanerophyte or N America & Neotropics DQ356143 DQ356209 AY350631 Chamaephyte 71 Lobelia leschenaultiana Nanophanerophyte or Southeast Asia DQ356131 DQ356196 Chamaephyte 72 Lobelia macrodon Hemicryptophyte Oceania EF694730 AY568742 73 Lobelia martagon Chamaephyte or Neotropics DQ356139 DQ356205 Hemicryptophyte 74 Lobelia nicotianifolia Nanophanerophyte or Southeast Asia AF042660 DQ285161 Herbaceous phanerophyte 75 Lobelia niihauensis Nanophanerophyte Hawaiian islands DQ285268 DQ285151 76 Lobelia nummularia Hemicryptophyte Southeast Asia DQ356164 DQ356235 77 Lobelia oligophylla Hemicryptophyte Neotropics DQ356159 DQ356227 78 Lobelia organensis Chamaephyte or Neotropics DQ285279 DQ285162 Hemicryptophyte 79 Lobelia perpusilla Hemicryptophyte Neotropics AY568741 80 Lobelia petiolata Phanerophyte Africa DQ285280 DQ285163 81 Lobelia physaloides Nanophanerophyte or Oceania AY568757+AY568745 Chamaephyte 82 Lobelia polyphylla Nanophanerophyte Neotropics DQ356123 DQ356177 AY350633 83 Lobelia portoricensis Chamaephyte or Neotropics DQ356142 DQ356208 Hemicryptophyte 84 Lobelia purpurascens Hemicryptophyte or Oceania DQ356160 DQ356229 Geophyte 85 Lobelia rotundifolia Nanophanerophyte Neotropics DQ356178 86 Lobelia roughii Hemicryptophyte Oceania DQ356165 EF126737 87 Lobelia sonderiana Hemicryptophyte or Africa DQ356130 DQ356195 Therophyte

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Table 3: List of species used in the analyses of the Lobelioideae data set, their life form and distribution (simplified from Lammers [23]), and GenBank accession numbers. (Continued)

88 Lobelia stricklandiae Phanerophyte Africa DQ356186 EU219381* 89 Lobelia stricta Hemicryptophyte Neotropics DQ356141 DQ356207 90 Lobelia tomentosa Chamaephyte Africa EF141028 DQ356180 91 Lobelia tupa Chamaephyte or Neotropics DQ356145 DQ356211 Hemicryptophyte 92 Lobelia villosa Nanophanerophyte Hawaiian islands DQ285293 DQ285176 93 Lobelia vivaldii Nanophanerophyte Neotropics DQ268873 DQ285167 94 Lobelia xalapensis Therophyte Neotropics DQ356144 DQ356210 95 Lobelia yuccoides Nanophanerophyte Hawaiian islands DQ356125 DQ356190 96 Lysipomia cuspidata Chamaephyte Neotropics DQ356133 DQ356198 AF054959 97 Lysipomia sphagnophila Chamaephyte Neotropics DQ356132 DQ356197 AF054943 98 Sclerotheca forsteri Nanophanerophyte French Polynesia DQ285287 DQ285170 99 Sclerotheca jayorum Nanophanerophyte or French Polynesia DQ285273 DQ285156 Phanerophyte 100 Siphocampylus affinis Nanophanerophyte or Neotropics DQ356155 DQ356223 Chamaephyte 101 Siphocampylus brevicalyx Nanophanerophyte Neotropics DQ356156 DQ356224 102 Siphocampylus fulgens Nanophanerophyte or Neotropics EF141032 DQ356216 Chamaephyte 103 Siphocampylus giganteus Nanophanerophyte or Neotropics DQ356154 DQ356222 Phanerophyte 104 Siphocampylus macropodus Nanophanerophyte Neotropics DQ356153 DQ356221 105 Siphocampylus scandens Nanophanerophyte Neotropics DQ356150 DQ356218 106 Solenopsis laurentia Therophyte Temp. Eurasia DQ356134 DQ356199 107 Trematolobelia kauaiensis Nanophanerophyte Hawaiian islands DQ285270 DQ285153 108 Trematolobelia macrostachys Nanophanerophyte or Hawaiian islands DQ356137 DQ356202 EU219382* Phanerophyte

Sequences produced in this study are marked with (*); their origin and voucher information are available from their GenBank record. relationship between Dendrosenecio and the silversword (as of August 2009). In order to assess which of these were alliance, and their separate diversification, has been gen- correctly placed in subfamily Lobelioideae, I first down- erally accepted for more than a century. This is reflected in loaded all Campanulaceae accessions for the most widely their taxonomic placement in different tribes based on used sequence regions: rbcL, trnL-F and ITS. Two criteria conspicuous floral dissimilarities and was recently corrob- were then used for choosing the ingroup taxa in order to orated by molecular phylogenetic analyses [42-45]. In infer relationships within subfamily Lobelioideae: (i) that contrast, the results presented here show a very different the species could be confidently assigned to the Lobelioi- evolutionary history for the giant form in the subfamily deae based on the results from the large-scale analyses for Lobelioideae. The giant lobelioids of Africa and the the Campanulaceae; and (ii) that the trnL-F region was Hawaiian Islands, together with similarly large species sequenced for that species, since this region has been from eastern Brazil, French Polynesia and southeast Asia, shown to contain considerably more phylogenetically are all derived from a single ancestor that was woody and informative characters than rbcL (41.7% as compared to probably African. 25.9%, respectively [21]). Based on these criteria, a total of 108 species (including seven outgroup species) were Detailed comparative studies are needed in order to eval- then selected for inferring the phylogeny of the Lobelioi- uate the morphological similarities and differences in deae. This represents an increase of 33 species compared habits among and within clades N4, N10 and N12 (Figure to the high-level phylogeny of the Lobelioideae recently 3). A different way of coding for the morphology and hab- presented by Antonelli [21]. In that analysis, sequences its of species in those clades might affect the reconstruc- for several taxa exhibiting a large/giant habit had not yet tion of the ancestral states for the Lobelioideae. been made available on GenBank. These include repre- Eventually, more phylogenetically informative sequence sentatives from the genera Apetahia and Sclerotheca (from data and denser taxon sampling may help elucidate the French Polynesia), Lobelia boninensis (from the Bonin evolution of this exceedingly diverse plant group. Islands), Lobelia sect. Galeatella (from the Hawaiian Islands) and Lobelia nicotianifolia (from southeast Asia), as Methods well as many other herbaceous species. Choice of taxa and molecular markers So far, some 2600 sequences classified as belonging to In an attempt to increase the phylogenetic resolution family Campanulaceae have been deposited in GenBank among representatives of the giant lobelioids, sequences

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Lobelia physaloides Lobelia coronopifolia Lobelia tomentosa Grammatotheca bergiana Lobelia aquatica Lobelia sonderiana Lobelia erinus Lobelia graniticola Lobelia anceps Lobelia columnaris Lobelia gibberoa Lobelia gregoriana Lobelia boninensis Lobelia petiolata Lobelia leschenaultiana Lobelia nicotianifolia Lobelia gloriamontis Lobelia kauaensis Lobelia villosa Apetahia longistigmata Apetahia margaretae Sclerotheca forsteri Sclerotheca jayorum Lobelia stricklandiae Lobelia exaltata Lobelia fistulosa Lobelia organensis Brighamia insignis Brighamia rockii Delissea rhytidosperma Delissea subcordata Delissea undulata Trematolobelia kauaiensis Trematolobelia macrostachys Lobelia niihauensis Lobelia hypoleuca Lobelia yuccoides Cyanea angustifolia Cyanea coriacea Cyanea kuhihewa Cyanea leptostegia Clermontia fauriei Clermontia arborescens Clermontia kakeana Clermontia parviflora Cyanea hirtella Cyanea acuminata Cyanea koolauensis Cyanea floribunda Cyanea pilosa Diastatea micrantha Solenopsis laurentia Downingia bacigalupii Downingia insignis Lobelia kalmii Lobelia rotundifolia Lobelia cardinalis Lobelia dortmanna Lobelia cordifolia Lobelia xalapensis Lobelia irasuensis Lobelia aguana Lobelia laxiflora Hippobroma longiflora Lobelia kraussi Lobelia martagon Lobelia portoricensis Lobelia stricta Lobelia vivaldii Lobelia excelsa Lobelia polyphylla Lobelia tupa Lobelia oligophylla Lobelia nummularia Lobelia macrodon Africa Lobelia roughii Isotoma axillaris French Polynesia Isotoma fluviatilis Lobelia purpurascens Hawaiian islands Lobelia chinensis Lobelia perpusilla North America Lobelia angulata Lobelia arenaria Neotropics Centropogon dissectus Centropogon luteus Oceania Centropogon trichodes Siphocampylus affinis Southeast Asia Siphocampylus brevicalyx Siphocampylus fulgens Temperate Eurasia Siphocampylus giganteus Node Absent Lysipomia cuspidata Lysipomia sphagnophila Equivocal Siphocampylus macropodus Siphocampylus scandens Centropogon cornutus Centropogon gamosepalus Centropogon granulosus Centropogon gutierrezii Burmeistera domingensis Burmeistera crispiloba Burmeistera cyclostigmata

50.0 40.0 30.0 20.0 10.0 0.0 AncestralFigure 9 range evolution in relation to time Ancestral range evolution in relation to time. Results from ancestral range reconstructions in subfamily Lobelioideae. Methodology as in Figure 7.

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The Partition Homogeneity Test [54] was applied to test for conflicting phylogenetic signal among these regions by performing a heuristic search with 5000 replicates, 100 random addition sequences, TBR branch swapping and saving up to 50 trees per replicate in PAUP* version 4.0b10 [55]. Since the test did not approach significance (P = 0.962), the three markers were combined for the sub- sequent analyses. A bootstrap analysis was run in PAUP under the maximum parsimony criterion, by sampling 10000 replicates, with 100 random addition sequences and saving one tree per replicate. A Bayesian phylogenetic analysis was then conducted with the software MrBayes v. 3.1 [56], performing two parallel runs of 20 million gen- erations each, using four chains, sampling every 1000 gen- erations and saving branch lengths. The performance of the analyses was evaluated using the software Tracer TinyFigure seeds 10 crossing long distances Tiny seeds crossing long distances. Lobelioid seeds are extremely small and could presumably be carried over large distances by strong wind currents. The figure shows seeds from the Hawaiian endemic Clermontia kakeana around the eye of a needle; one gram contains about 36,000 seeds. of the ITS (ITS 1 - 5.8S - ITS 2) of nuclear ribosomal DNA were generated de novo for 10 giant species plus Lobelia dortmanna. The region was amplified and sequenced with the primers ITS1, ITS2, ITS3, ITS4 [46], and ITS10 [21], following the amplification and sequencing techniques described by Antonelli [21]. Since ITS is one of the fastest evolving molecular markers available today [47-49], its use was expected to add resolution for identifying the rela- tionships between the main groups of giant lobelioids. Table 3 lists all species used in the analyses of subfamily Lobelioideae and indicates the species sequenced for this study.

Alignment and phylogenetic estimation Sequences were aligned using the L-INS-I algorithm implemented in the software MAFFT v. 6 [50]. Aligned matrices were inspected manually and all unreliable sequences excluded, iteratively, until all sequences were deemed homologous. The final aligned matrices for Cam- panulaceae comprised: for trnL-F, 452 sequences and 1541 characters; for ITS, 445 sequences and 1197 charac- ters; and for rbcL, 438 sequences and 1401 characters. LargeFigure habit 11 attained elsewhere Large habit attained elsewhere. Although truly giant Maximum Likelihood trees were inferred in the software lobelioids (with a thick stem and a large terminal leaf rosette) Garli 0.960 [51] by performing 10 independent runs with all belong to the same clade, some Neotropical species in the the default settings. The analyses were performed in the genera Siphocampylus, Centropogon and Burmeistera can be CIPRES cluster at the San Diego Supercomputer Center rather tall shrubs. The Andean Siphocampylus giganteus por- http://www.phylo.org/portal2 and the results visualized trayed here is one example which has been coded as varying using the Interactive Tree of Life facility http:// between nanophanerophyte and phanerophyte. [Credit: Len- itol.embl.de. MrModeltest [52,53] was used to select the nart Andersson]. best-fitting evolutionary model for each sequence region.

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v.1.4.1 [57] and node frequency statistics calculated on of 5.33 Ma for subfamily Campanuloideae. (iii) A 29.8 20000 post burn-in trees. Ma maximum age was imposed for the radiation of the endemic Hawaiian taxa. This corresponds to the age of the Molecular dating oldest island of the Hawaiian Ridge (Kure), after which a Several methods are currently available for estimating continuous chain of islands has been available for prop- divergence times in a phylogeny [58,59]. In order to com- agules of the highland Hawaiian biota. In the PL analysis, pare the results from two widely used methods, diver- this provided minimal ages for the five strongly supported gence times were estimated using the Bayesian relaxed (Bpp = 1.00) crown groups of Hawaiian species identified clock and PL approaches [60,61]. Both methods have the in the MrBayes analysis (see Figure 4, Results). In order to advantage of enabling direct calibration on one or more be able to use the latter as a calibration point in the BEAST nodes of a phylogeny. A major difference between them, analyses, it first was necessary to constrain all Hawaiian however, is that PL assumes that rates are auto-correlated species to be monophyletic (failure to do so caused the (inheritable), whereas in Bayesian relaxed clock dating program to crash, apparently due to a failure in generating each branch is allowed to evolve at its own rate. starting trees). Since the monophyly of the Hawaiian lobelioids was demonstrably strongly supported [Bpp = PL estimations were done using the software r8s [61,62]. 1.00, 8, 10], this a priori constraint was not expected to sig- An automated cross-validation algorithm was run to iden- nificantly influence the results. tify the optimal smoothing value for the final analysis, with log10 increments of 0.1 and using the Truncated Biogeographic and character evolution analyses Newton method implemented in r8s. For estimating the One problematic aspect in referring to 'giant lobelioids' is effects of phylogenetic uncertainty on node age estima- how we should characterize these plants morphologically. tions, 1000 trees from the stationary sample of Bayesian Giant lobelias have been described as: trees were independently dated and their statistics (mean and 95% confidence interval values) computed for each 'pachycaul plants; often long-lived and pliestesial; of large node of the Bayesian consensus tree using the software stature and bulk; with dense apical rosettes of typically TreeAnnotator [63]. sessile leaves, which may close every night and are retained even after they die for insulation and fire protec- For the Bayesian dating analysis, five runs of 10 million tion; with nightly closing, large racemose inflorescences; generations each were performed in the software BEAST with a large apical bud of developing leaves to protect v.1.4.8 [22] at the Computational Biology Service Unit young tissue; which secrete ice-nucleating polysaccharide hosted by Cornell University, USA http://cbsuapps.tc.cor fluids that prevent frost damage; and a hollow pith for nell.edu. The analysis assumed a pure birth (Yule) proc- internal water storage that narrows basipetally'. (see [7] ess, since this tree prior is most suitable for inferring rela- and references therein [23,69,70]). tionships between individuals from different species [22]. The performance of the analysis (convergence of the inde- These features correctly describe several of the eastern Afri- pendent runs, effective sample sizes) was evaluated using can species but they do not apply to all of them, let alone Tracer v.1.4.1 [22]. Mean and 95% HPD intervals of ages to other large lobelioids elsewhere (for example, the were then calculated from 40,000 post burn-in trees using Andean species Siphocampylus giganteus, Figure 11). To the software TreeAnnotator v.1.4.8 [22], and visualized overcome this problem, life form was coded here accord- using FigTree v1.2.2 [63]. ing to the recent checklist of the Campanulaceae by Lam- mers [23] (Table 2). Besides being a comprehensive and Three simultaneous calibration points were applied in up-to-date account on life form and distribution for all both analyses. (i) Since the fossil record of Campanu- species, this work provides a consistent framework for laceae is exceptionally scarce [64], the root of the Asterales evaluating evolutionary traits in the family. The current tree (see [65]) was calibrated at 93 Ma, as estimated under distribution of all species was compiled from the same a major study including 83 asterid families and based on source [23], but simplified into eight larger operational six plastid markers and six fossil calibrations [66]. Relia- units: Africa, French Polynesia, the Hawaiian Islands, tem- bility of this age was recently corroborated by a study that perate North America, the Neotropical region including employed the same set of fossils, but a different set of temperate South America, Oceania, southeast Asia and sequence regions and taxa (mean 94 Ma; [67]). In the PL temperate Eurasia. For both characters, the outgroup analysis, 93 Ma was set as a fixed calibration, whereas in taxon (Helianthus) was coded as having a different state to the BEAST analyses it constituted a normally distributed any of the ingroup taxa, to prevent biasing the reconstruc- prior with mean 93 Ma and standard deviation of 1.0. (ii) tion of basal nodes on the tree (since Helianthus is used An unchallenged fossil Campanula from the Miocene of here to root the Campanulaceae tree but it is certainly not Poland [68] was used to impose a minimal age constraint

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the sister species to the Campanulaceae). Life form and Abbreviations distribution for all species analysed are listed in Table 3. BEAST: Bayesian evolutionary analysis by sampling trees; Bpp: Bayesian posterior probability; Bs: Bootstrap sup- The most widely used methods of ancestral area recon- port; CI: Confidence interval; HPD: Highest Posterior struction that are able to take into account phylogenetic Density; ITS: internal transcribed spacer; Ma: Mega- uncertainty and multistate characters are Fitch Parsimony annum (million years); MRCA: most recent common (FP), implemented in the software Mesquite v. 2.7 [71] ancestor; PL: Penalized Likelihood; SCBL: Siphocampylus, and Bayes-DIVA [72,73]. These two methods are based on Centropogon, Burmeistera, Lysipomia. very different biogeographic evolutionary models. FP con- strains ancestors to be monomorphic (that is, restricted to Acknowledgements single areas) and models changes in distribution from I would like to thank Roger Eriksson, Claes Persson, Aelys Humphreys, ancestor to descendant as a change in character state, Bengt Oxelman, Thomas Lammers, Eric Knox, Uno Eliasson, Derrick equivalent to dispersal between single areas. It thus imple- Zwickl and Toby Pennington for fruitful discussions and advice, Vivian ments a dispersalist explanation. In contrast, DIVA allows Aldén for laboratory assistance, and Gerald Carr, Frederick Warshauer and Christian Puff for kindly providing the photographs in Figure 1. Thomas widespread distributions at ancestral nodes. Although the Givnish and three anonymous reviewers are kindly acknowledged for maximum number of areas can be constrained in DIVA, reviewing and providing constructive criticism on this manuscript. This single-area ancestors are not allowed and widespread dis- research has been financially supported by grants from the Royal Swedish tributions are always divided at speciation events by vicar- Academy of Sciences, the Swedish Research Council to Claes Persson, the iance. Thus DIVA favours a more vicariant explanation Royal Society of Arts and Sciences in Göteborg, Kungliga och Hvitfeldtska [74]. Based on the molecular dating results obtained (see Stiftelsen, Helge Ax:son Johnsons Stiftelse, Carl Tryggers Stiftelse and Uni- Results and Discussion), and taking into consideration versity of Gothenburg. that the Hawaiian archipelago has never been connected to any land mass [26], it is clear that long-distance disper- References 1. Linnaeus C: Systema Naturae Volume 1. 10th edition. Laurentii Salvii, sals have played a crucial role in shaping the distribution Holmiae; 1758:824. of the Lobelioideae. This indicated that Fitch optimiza- 2. Givnish TJ, Sytsma KJ: Molecular Evolution and Adaptive Radi- ation. Cambridge: Cambridge University Press; 1997. tion was a more suitable method for inferring ancestral 3. Futuyma DJ, Edwards SV, John R: Evolution. Massachusetts: Sinauer ranges of lobelioid nodes than DIVA. Mesquite v. 2.7 [71] Associates; 2005. was therefore used for reconstructing both ancestral areas 4. Hedberg O: Features of afroalpine plant ecology. Stockholm: Almqvist & Wiksell; 1964. and ancestral life forms. To take into account phyloge- 5. Fries RE, Fries TCE: Die riesen-lobelien Africas. Svensk Botanisk netic uncertainty, reconstructions were performed on Tidskrift 1922, 16:383-416. 1000 trees from the stationary Bayesian tree sample, using 6. Mabberley DJ: The giant lobelias: pachycauly, biogeography, ornithophily and continental drift. New Phytologist 1975, the Maximum Parsimony criterion and counting all trees 74:365-374. with uniquely best states. The results were summarized by 7. Knox EB, Palmer JD: Chloroplast DNA evidence on the origin and radiation of the giant lobelias in eastern Africa. Systematic computing the relative frequencies of ancestral area recon- Botany 1998, 23:109-149. structions for each node of the Bayesian consensus clado- 8. Givnish TJ: Adaptive radiation, dispersal, and diversification of gram of the Lobelioideae. the Hawaiian lobeliads. In The biology of biodiversity Tokyo: Springer; 1999:67-90. 9. Givnish TJ, Sytsma KJ, Smith JF, Hahn WJ: Thorn-like prickles and Tests of phylogenetic conservatism heterophylly in Cyanea: Adaptations to extinct avian brows- In order to test whether life form and geographic distribu- ers on Hawaii? Proceedings of the National Academy of Sciences of the United States of America 1994, 91:2810-2814. tion were phylogenetically conservative, I first calculated 10. Givnish TJ, Millam KC, Mast AR, Paterson TB, Theim TJ, Hipp AL, the number of parsimony steps necessary to explain the Henss JM, Smith JF, Wood KR, Sytsma KJ: Origin, adaptive radia- tion and diversification of the Hawaiian lobeliads (Asterales: occurrence of each character on a particular tree, repeating Campanulaceae). Proceedings of the Royal Society B: Biological Sci- this for 1000 trees randomly chosen from the post burn- ences 2009, 276:407-416. in Bayesian sample (the same sample used for the dating 11. Lammers TG: Chromosome numbers of Campanulaceae. III. Review and integration of data for subfamily Lobelioideae. and character state reconstruction analyses). I then gener- Amer J Bot 1993, 80:660-675. ated 1000 trees with the same topology as the Bayesian 12. Lammers TG, Hensold N: Chromosome numbers of Campanu- laceae. II. The Lobelia tupa complex of Chile. Amer J Bot 1992, 50% majority-rule consensus but with character states 79:585-588. randomly shuffled among the tips of each tree, while 13. Knox EB: The conservation status of the giant senecios and keeping their relative frequency constant. The values giant lobelias in eastern Africa. Opera Botanica 1993, 121:195-216. obtained after reconstructing the characters on the simu- 14. Embuscado ME, BeMiller JN, Knox EB: A survey and partial char- lated data set were used to compute 99% credibility inter- acterization of ice-nucleating fluids secreted by giant-rosette vals, to which the observed numbers of parsimony steps (Lobelia and Dendrosenecio) plants of the mountains of eastern Africa. Carbohydrate Polymers 1996, 31:1-9. for each respective character could be compared. The anal- 15. Givnish TJ, Montgomery RA, Goldstein G: Adaptive radiation of yses were performed in Mesquite v. 2.7 [71]. photosynthetic physiology in the Hawaiian lobeliads: Light

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