Phylogenetic Reticulation in Subtribe Helianthinae Author(s): Edward E. Schilling and Jose L. Panero Source: American Journal of Botany, Vol. 83, No. 7 (Jul., 1996), pp. 939-948 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2446272 . Accessed: 26/12/2010 17:01

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http://www.jstor.org AmericanJournal of Botany 83(7): 939-948. 1996.

PHYLOGENETIC RETICULATION IN SUBTRIBE HELIANTHINAE1

EDWARD E. SCHILLING2 AND JOSE L. PANERO3

Departmentof Botany,University of Tennessee, Knoxville, Tennessee, 37996-1100; and Departmentof Botany and Pathology,Michigan State University,East Lansing, Michigan 48824-1312

Incongruencebetween phylogenetic estimates based on nuclearand chloroplastDNA (cpDNA) markerswas used to infer that there have been at least two instances of chloroplasttransfer, presumably through wide hybridization,in subtribe Helianthinae. One instance involved dombeyana, which exhibited a cpDNA restrictionsite phenotypethat was markedlydivergent from all of the otherspecies of the thatwere surveyedbut thatmatched the restrictionsite pattern previouslyreported for South Americanspecies of .In contrast,analysis of sequence data fromthe nuclear ribosomal DNA internaltranscribed spacer (ITS) region showed Simsia to be entirelymonophyletic and placed samples of S. dom- beyana as the sistergroup to the relativelyderived S. foetida, a resultconcordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sistergroup to a memberof V. subg. Amphilepis,which was consistentwith cpDNA restrictionsite data. Samples of Tithoniaformed a single monophyleticclade based on ITS sequence data, whereasthey were splitbetween two divergentclades based on cpDNA restrictionsite analysis. The results suggested that cpDNA transferhas occurred between taxa diverged to the level of morphologicallydistinct genera,and highlightthe need forcareful and completeassessment of moleculardata as a source of phylogeneticinformation.

Key words: chloroplastDNA (cpDNA); Helianthinae;nuclear ribosomal DNA internaltranscribed spacer (ITS); phy- lopeneticreticulation.

Study of phylogeneticreticulation is of particularin- tected,the competinghypothesis that the incongruenceis terestbecause the frequencyof naturalhybridization is the resultof homoplasious changes must also be consid- one of the most distinctiveyet poorly understoodele- ered, and thisis particularlyso when functionaltraits that mentsof floweringplant diversity (e.g., Grant,1981). De- could be subject to selective pressuresare used. The po- spite considerableinterest, the ultimateevolutionary sig- tentialfor homoplasy in such situationsis enhanced by nificanceof naturalhybridization remains uncertain.At the fact thatgroups close enough geneticallyto produce one end of the spectrum,it is possible thatnew genetic hybridswhen crossed will necessarilytend to have sim- combinationsproduced throughhybridization may lead ilar morphologicalor biochemical potentials.Lineage or to novel divergence,and thus be of evolutionarysignif- allelic sorting(Doyle, 1992) is yet anotheralternative hy- icance. In contrast,it has also been hypothesizedthat hy- pothesisthat may account forincongruence between data bridization is an "evolutionary sideshow," producing sets, particularlybetween gene . temporaldiversity that may be strikingbut has no trace- Detection of phylogeneticreticulation has been en- able long-termeffects. Phylogenetic reticulation may also hanced greatlyby the availabilityof methodsto analyze be of significanceas a potentialsource of errorfor clas- DNA variationdirectly. Molecular data are readily sub- sificationsystems that are based partlyor solely on in- divided into discrete sets, such as those for individual ferredphylogeny and thatemploy models of divergence genes, that still offerenough variantcharacter states to thatare strictlydichotomous. resolve phylogenetichypotheses. Many or most molec- Assessmentof the evolutionaryimportance of natural ular variantsmay be selectively neutral.Rieseberg and hybridizationis complicatedby the factthat the evidence Soltis (1991) summarize 36 examples of chloroplast forphylogenetic reticulation is based on patternsof char- DNA (cpDNA) "capture" involving 17 genera. A num- acter distributionswhose detectionis difficultand which ber of these examples involve cases in which phyloge- can be explained by alternativehypotheses. Evidence for netic reticulationhad not been anticipatedfrom other phylogeneticreticulation comes primarilyfrom incongru- types of data. ence of phylogeneticestimates based on differentchar- Detection of phylogeneticreticulation does not ensure acter sets. The existenceof discretesubsets of characters thatit is of evolutionarysignificance, but it does forma that are incongruentmay be overlooked when morpho- necessary foundationfor further examination of the phe- logical or chemical data are employed. Even when de- nomenon. In this respect,the documentationthat phylo- genetic reticulationhas occurred in a group prior to its 1 Manuscript received 3 May 1995; revision accepted 26 January. subsequentdivergence would be of particularimportance 1996. The authorsthank P. B. Cox and M. L. Schmid for technicalassis- by providingthe possibilitythat genetic materialtrans- tance, J. La Duke and D. Spooner for plant samples, and the staffof ferredthrough hybridization had alteredthe evolutionary MEXU for logistical support.Automated fluorescentsequencing was potentialof the group. Many of the examples cited by performedby the MSU-DOE-PRL Plant BiochemistryFacility using Rieseberg and Soltis (1991) involve single species within the ABI Catalyst 800 for Taq cycle sequencing and the ABI 373A a genus, or closely related species where the reticulation Sequencer forthe analysis of products.This researchwas supportedby may not differ substantiallyfrom regular gene flow NSF grantsDEB-9019158 to EES and DEB-9496174 to JLP. 2 Authorfor correspondence. throughoutcrossing. A notable exception is an example 3Current address:Department of Botany,University of , Austin, involvingintergeneric cpDNA transferthat was reported Texas 78713-7640. by Soltis et al. (1991) fromSaxifragaceae. 939 940 AMERICAN JOURNAL OF BOTANY [Vol. 83

SubtribeHelianthinae has been the subject of several TABLE 1. List of samples of Helianthinaeused for chloroplastDNA previousmolecular studies,and phylogeneticreticulation restrictionstudies or for sequence studies of the nuclearribosomal internaltranscribed spacer region. * sample newly analyzed for in the subtribehas been documentedwithin a single sec- chloroplastDNA data. tion of one genus. The subtribeis a memberof the As- teraceae, tribeHeliantheae, and is well definedmorpho- Genus and logically (Robinson, 1981; Bremer,1994). Results of sur- sample number Species Geographicalorigin veys of cpDNA restrictionsite variationhave added ev- idence that the subtribe is monophyletic (Schilling, 1001 H. multiflora USA Panero, and Eliasson, 1994). The cpDNA studies have Pappobolus 720 P. lehmannii also helped to confirmthat Viguiera, the core genus of Simsia the subtribe,is paraphyletic,and to refinethe circum- 4223*, 4230*, scriptionof, and suggest relationshipsfor, infrageneric 4272* S. amplexicaulis groups of the genus (Schilling and Jansen,1989; Schil- 443*, 2416* S. annectens Mexico 84-1 S. calva USA ling and Panero, 1991). Detailed studies of Helianthus 193*, 306* S. dombeyana ,Ecuador sect. Helianthus have shown that phylogeneticreticula- 2434*, 2699 S. foetida Mexico, tion has been a significantevolutionary factor in thisdip- 1099* S. fruticulosa Ecuador loid, annual group (Rieseberg, Carter,and Zona 1990; 1803* S. ghiesbreghtii Mexico Beckstrom-Sternberg,and Doan, 1990; Rie- 2268* S. lagascaeformis Mexico Rieseberg, 2406*, 2468* S. sanguinea Mexico seberg, 1991; Rieseberg et al., 1991; Rieseberg and 88-43* S. spooneri Mexico Brunsfeld,1991). A broadersurvey has failed, however, 88-35* S. sylvicola Mexico to detectany evidence of intersectionalreticulation in the 2789* S. villasenorii Mexico genus (E. E. Schilling,unpublished data). 2213*, 2862* T. calva Mexico Although application of data from molecular studies 2452* T. diversifolia Mexico has greatlyadvanced our understandingof phylogenetic -* T. koelzii Mexico relationshipsin subtribeHelianthinae, some puzzling re- 2523* T. longiradiata Mexico sults have been obtained.Among these is the suggestion 2505* T. pedunculata Mexico of a close relationshipbetween Tithonia and Simsia 2395*, 2510*, 2566 T. rotundifolia Mexico (Schilling and Jansen, 1989), which are each relatively 295* T. thurberi USA distinctivemorphologically (LaDuke, 1982a; Spooner, 2373*, 2417* T. tubaeformis Mexico 1990). Cladistic analysis of morphologicaldata (Schilling Viguiera and Panero, 1991; Panero, 1992) has notbeen particularly subg. Amphilepis 2447* V. angustifolia Mexico illuminatingwith regardto this or otherproblematic hy- 2383* V. buddleiformis Mexico potheses of relationshipsin Helianthinae based on cp- 2781*, 2869* V. ensifolia Mexico DNA data. This has made it highlydesirable to seek data 2304*, 2390*, fromother sources, particularlyfrom a nuclear-encoded 2391* V. excelsa Mexico gene region,and led to the currentstudy. 88-51 V. flava Mexico 2335*, 2399*, Here we presentdata to suggest thatphylogenetic re- 2454* V. hemsleyana Mexico ticulationinvolving clades recognized as distinctgenera 2421*, 2560* V. hypochlora Mexico has occurredwithin subtribe Helianthinae. The evidence 2443* V. pachycephala Mexico involves incongruencebetween data fromcpDNA restric- 2449* V. schultzii Mexico subg. Bahiopsis tion site analysis and sequence analysis of the internal 18 V. tomentosa Mexico transcribedspacer region (ITS) of the nuclear ribosomal Subg. Viguiera DNA (rDNA). For one case, involvingSimsia dombey- sect. Leighia ana, the evidence forreticulation seems quite convincing 88-46* V. linearis Mexico because the cpDNA data also are in conflictwith clear- sect. Maculatae 15298 V. puruana Mexico cut morphologicalobservations. In the second case, in- sect. Paradosa volving species of Tithonia,it appears that divergence 863 V. szyszylowiczii has occurred subsequent to the reticulationevent. The sect. Viguiera resultsof thisstudy suggest that phylogenetic reticulation ser. Grammatoglossae 1814* V. benziorum Mexico is a phenomenonthat must be consideredin the circum- 2595* V. bombycina Mexico scriptionand analysis of relationshipsof genera. 2581* V. davilae Mexico 2832 V. grammatoglossa Mexico MATERIALS AND METHODS 621, 88-36* V. ovata Mexico 2313* V. purpusii Mexico Sample of subtribeHelianthinae and near outgroups(Table 1) were 2439* V. parkinsonii Mexico grown in the Universityof Tennessee greenhouses from achenes or 2861* V. pringlei Mexico rhizomes,or were collected in the fieldin liquid N2 and storedat -80?C 88-28*, 31*, Mexico untilready forDNA extraction.Preparations of total DNA were made 39*, 40* V. rhombifolia 2780* V. seemannii Mexico fromfresh (0.5-2.0 g) or frozen (1-3 g) by the procedureof 85-25, 2385* V. sessilifolia Mexico Doyle and Doyle (1987). ser. Pinnatilobatae Methods for DNA restrictionsite analysis generally followed Jan- 85-28, 2502 V. pinnatilobata Mexico sen and Palmer (1988). Restrictionendonuclease digestion;s(utiliz- OUTGROUP ing the following enzymes: AvaI, BamHI, BanI, BanlI, BclI, BglII, Flourensia Mexico BstNI, BstXI, ClaI, EcoRI, EcoRV, HaeII, HincII, HindIII, NcoI, 2165 F. monticola July 1996] SCHILLING AND PANERO-PHYLOGENETIC RETICULATION IN HELIANTHINAE 941

NsiI), agarose gel electrophoresis,and bidirectionaltransfer of DNA consistency index of 0.84 (0.92 with autapomorphies fragmentsfrom agarose gels to Zetabind (AMF CUNO, Meriden, included), a retentionindex of 0.97, and a rescaled con- CT), Hybond (Amersham, Arlington Heights, IL), or Boehringer- sistency index of 0.90. The distributionsand numbers Mannheim (Indianapolis, IN) nylon filterswere performedas de- of characterchanges and relative values of supportdid scribed in Palmer (1986) and Jansen and Palmer (1987). For some not differsignificantly from trees reported earlier for sets of samples, preparationof digoxigenin-labeled probes and filter subtribe Helianthinae (Schilling and Jansen, 1989; hybridizationsfollowed the manufacturer'sinstruction (Boehringer- Schilling, Panero, and Eliasson, 1994). Mannheim, "Genius Kit," Indianapolis, IN). For most samples, The newly reported samples for cpDNA data were preparationof 32P-labeled probes and filterhybridizations followed placed by parsimonyanalysis in the two terminalclades Palmer (1986), except that random priming ratherthan nick-trans- of Helianthinae (Fig. 1). Samples of Simsia and some The 22 cloned lation methodology was employed to label probes. herbaceous membersof Viguiera ser. Grammatoglossae fragmentsof lettuce cpDNA were combined into batches restriction togetherin a single large clade. Samples of for filterhybridization experiments. Mapping of fragmentswas done were placed relative to data of Jansen and Palmer (1987, 1988), Schilling and S. sanguinea, S. foetida, and S. amplexicauliswere united Jansen (1989), and E. E. Schilling (unpublished data). In most gel by fourapomorphies, but otherwise,there was littleres- runs, fragmentscould be detected to a lower limit of about 400 bp. olutionwithin the clade. Samples of one otherspecies of Sequencing of the ITS region followed standardprotocols (Baldwin, Simsia, S. dombeyana,were placed by cpDNA resultsin 1992), except that an automated sequencer was employed. Single- the clade containingthe South American species of Vi- strandedDNAs of the entireITS/5.8S region were amplifiedby the guiera and were separatedfrom other members of Simsia polymerasechain reaction (PCR) using the primersthat flankthis re- by a minimumof 25 restrictionsite changes. Samples of gion. The PCR productswere resolvedby electrophoresisin 3% agarose V. subg. Amphilepiswere placed in the second major ter- minigels,cut out, redissolved,and subsequentlypurified. The ITS se- minal clade of Helianthinae,as sisterto the group con- quences in Helianthinae were sufficientlysimilar to one anotherthat taining the South American species of Viguiera. Also theycould be aligned readilyby eye. placed in this second clade were the remainingsamples For cpDNA data, Flourensia and were used as outgroupsto of V. ser. Grammatoglossae,which were primarilythe polarize characterchanges withinHelianthinae. Wagner parsimony anal- woody ones. Samples of Tithoniawere placed in each of ysis was performedfor restriction site data utilizingthe PAUP program the two derivedHelianthinae clades (Fig. 1). Samples of (Swofford,1993). Most parsimonioustrees were soughtby implement- T. diversifolia,T. koelzii, T. rotundifolia,and T. thurberi search option,using the TBR branch-swapping ing the generalheuristic were placed near those of Simsia, but differedfrom them MULPARS turnedon. option and with site change. Samples of For the ITS sequence data, the data matrixwas enlargedto include by lack of a single apomorphic sequences fromArnica mollis (Baldwin, 1992) and T. calva, T. longiradiata, T. pedunculata, and T. tubae- (L. R. Rieseberg,unpublished data), representingan additionaloutgroup formisformed a well-supportedgroup (four apomorphic and anothermember of Helianthinae,respectively. The data matrixwas site changes) thatwas sisterto the groupcontaining sam- analyzed by Wagnerparsimony using PAUP, version 3.1.1 (Swofford, ples of V. ser. Grammatoglossaeand subg. Amphilepis. 1993). Only those nucleotidesites with unambiguousalignments were Two furtherPAUP searches were done, with the con- used in the phylogeneticanalysis, and gaps were treatedas missingdata. straintsthat eitherSimsia or Tithonia be monophyletic, The general heuristicsearch option of PAUP, with the TBR branch- respectively.With the constraintthat all species of Simsia swapping option,MULPARS on, and collapse of zero-lengthbranches, be monophyletic,the lengthof the shortesttree was 197 was used to search for maximallyparsimonious trees. Consensus trees steps, an increase of 25 steps (14% longer). The con- were calculated using the strictconsensus option. Following the decay straintthat all species of Tithonia be monophyleticre- analysis approach of Donoghue et al. (1992), -lengthconstraints sulted in a shortesttree of 189 steps, an increase of 17 were used to compile sets of trees up to three steps longer than the steps (10%). maximally parsimoniousones to determinethe numberof additional steps required to collapse sister-grouprelationships in at least one of the shortesttrees. Bootstrap values (Felsenstein, 1985) for particular Features of ITS in Helianthinae-The organization clades were calculated from 100 replicateWagner parsimony analyses and relativelengths of the ITS-1, ITS-2, and 5.8S regions using the PAUP "heuristics" option and "closest" addition sequence were very similar to those reportedby Baldwin (1992, of the taxa. 1993) forother members of tribeHeliantheae. The entire ITS region varied in lengthamong samples of Helianthi- RESULTS nae from 649 to 655 base pairs (bp). ITS-1 ranged in Chloroplast DNA restriction site analysis-In ad- lengthfrom 261 to 265 bp and ITS-2 from224 to 228 dition to some of the 183 variants noted previously bp. The 5.8S subunitwas uniformin size (164 bp) among (Schilling and Jansen, 1989; Schilling and Panero, all of the samples. 1991; Schilling, Panero, and Eliasson, 1994), 38 novel Alignmentof sequences of Helianthinaeto one another cpDNA restrictionsite variants were revealed among requiredgaps at 4.9% of positionsin ITS-1 and 6.6% of the newly analyzed samples of subtribe Helianthinae positionsin ITS-2. Alignmentof sequences to the nearest (Table 2). Results of cladistic analyses suggested that outgroup,Flourensia, required additional gaps. Align- most of the novel site changes representedautapomor- ment of Flourensia and Helianthinaerelative to another phies for individual samples or groups that had not outgroup,Arnica mollis, requiredadditional gaps. been examined previously. For this study, a data set Levels of divergencein ITS between samples within that included 50 species scored for the presence or ab- Helianthinaeranged from0.4 to 13.0%. Divergence lev- sence of 156 variable restrictionsites was analyzed. els for samples of Helianthinaeto Flourensia monticola Parsimony analysis resulted in a single most parsimo- were higher,with an average of 16.8% for all pairwise nious tree (Fig. 1), which had a length of 170 steps, a comparisons. Divergence levels between Helianthinae 942 AMERICAN JOURNAL OF BOTANY [Vol. 83

TABLE 2. ChloroplastDNA restrictionsite variantsobserved in samples of subtribeHelianthinae. Characters 1-183 as in Schilling and Jansen (1989), Schilling and Panero (1991), and Schiling,Panero, and Eliasson (1994). Probes fromlettuce Sacl cpDNA clone library,listed by size. Ancestral fragmentor fragmentsare listed to left of equal sign. Small fragmentsinferred, but not visualized directly,are listed in square brackets.Sample numbers(see Table 1) listed where not all samples of a species exhibitedthe site change.

No. Probe Enzyme Fragments(in KB) Samples 184 18.8 BclI 3.5 + 1.8 = 5.3 V. pringlei,V. seemannii 185 18.8 NcoI 11.0 = 6.6 + 4.4 T. tubaeformis 186 14.7 AvaI 4.6 = 3.3 + 1.3 V. pringlei,V. seemannii 187 14.7 BamHI 2.2 + 1.6 = 3.8 T. calva, T. longiradiatia,T. pedunculata, T. tubaeformis 188 14.7 BamHI 0.8 = 0.7 [+0.1] T. rotundifolia(2566) 189 14.7 BglII 3.7 = 2.8 + 0.9 T. rotundifolia(2510) 190 14.7 BstNI 1.1 + 0.8 = 1.9 T. thurberi 191 14.7 EcoRV 7.8 = 5.0 + 2.8 S. foetida 192 14.7 HaeII 2.4 + 6.3 = 8.7 T. thurberi 193 14.7 HincII 3.4 = 3.3 [+0.1] T. calva (2213) 194 14.7 HincII 3.0 + 3.4 = 6.4 T. pedunculata, T. thurberi 195 14.7 NsiI 3.7 + 5.9 = 9.5 T. thurberi 196 12.3 BanIl 8.0 = 4.4 + 3.6 S. ghiesbreghtii 197 10.6 BanI 7.4 = 6.4 + 1.0 T. tubaeformis 198 10.6 EcoRI 3.7 = 3.4 [+0.3] T. tubaeformis(2417) 199 10.6 EcoRV 5.8 = 2.9 + 2.9 V. buddleiformis(2383) 200 10.6 NsiI 1.8 + 1.0 = 2.8 S. annectens(2416) 201 9.9 Bcll 15.0 + 2.6 = 17.6 S. amplexicaulis (4230) 202 7.7 BglIl 4.6 = 3.0 + 1.6 T. longiradiata 203 7.7 BstNI 5.3 = 3.5 + 1.8 T. calva, T. longiradiatia,T. pedunculata, T. tubaeformis 204 7.0 BanI 3.5 = 3.0 + 0.5 V. angustifolia 205 7.0 BstNI 3.6 = 2.8 + 0.8 V. angustifolia,S. ghiesbreghtii 206 7.0 BstXI 5.8 + 1.7 = 7.5 S. amplexicaulis 207 7.0 EcoRV 4.6 + 0.6 = 5.2 S. amplexicaulis 208 6.9 BstXI 3.2 + 9.4 = 12.6 S. dombeyana 209 6.7 Bcll 6.7 + 5.6 = 12.3 V. excelsa, V. flava, V. hypochlora,V. pachy- cephala 210 6.7 BstXI 5.8 + 1.2 = 7.0 S. amplexicaulis (4223) 211 6.7 ClaI 9.1 = 6.1 + 3.0 V. excelsa, V. flava, V. hypochlora,V. pachy- cephala 212 6.7 NsiI 16.7 = 10.7 + 6.0 T. calva (2213) T. rotundifolia(2395) 213 5.4 BanI 3.6 + 3.0 = 6.6 T. calva (2213) 214 5.4 BstNI 2.0 = 1.2 + 0.8 T. hemsleyana(2399) 215 5.4 ClaI 5.2 [+0.5] = 5.7 T. tubaeformis(2417) 216 4.6 AvaI 8.5 + 4.9 = 13.4 T. tubaeformis 217 4.6 BstNI 7.4 = 6.4 + 1.0 T. thurberi 218 4.6 ClaI 1.8 + 0.5 = 2.3 V. parkinsonii(2439) 219 4.6 HaeII 5.7 = 4.8 + 0.9 T. tubaeformis 220 3.8 HincIl 3.0 + 4.6 = 7.6 T. calva, T. longiradiatia,T. pedunculata, T. tubaeformis 221 pet BglII 3.7 = 2.8 + 0.9 T. rotundifolia(2510) samples and Arnica mollis were also higher,with an av- ITS sequence sites in Helianthinaeand outgroupsamples. erage of 24.2%. These trees had a lengthof 422 steps and a consistency A charactermatrix of 503 characterswas necessaryto index of 0.59, excluding uninformativesites. The strict align Helianthinaeand outgroupITS DNA sequences. Of consensus of these 12 trees is presentedin Fig. 2. One these 503 characters,229 (45.5%) were variable. ITS-1, of these 12 trees is shown in Fig. 3 to indicate numbers with52% of sites variable,was more variablethan ITS-2, of point mutationssupporting each clade. which had 38% of sites variable. Potentiallyinformative A decay index analysis showed thatcollapse of branch- charactersaccounted for 19% of all ITS sites and 42%. es in the strictconsensus tree required one to three or of variable ones. more additional steps (Fig. 2). Bootstrap values for the Twelve minimum-lengthtrees were generated from consensus clades ranged from28% to 99% (Fig. 2). Wagner parsimony analysis of potentiallyinformative Constraintanalyses were performedusing PAUP to as-

Fig. 1. Single most parsimoniouscladogram showing relationshipsamong samples of Viguiera and othermembers of subtribeHelianthinae based on chloroplastDNA restrictionsite data. Numbers of site changes shown above branches; bootstrapvalues (out of 100 replicates) shown below branches. July 1996] SCHILLING AND PANERO-PHYLOGENETIC RETICULATION IN HELIANTHINAE 943

Flourensia

10 - V. tomentosa subg. Bahiopsis Heliomeris multiflora V. puruana sect. Maculatae Helianthus annuus 85 Pappobol us 1 ehmannii

V. pinnatilobata sect. Pinnatilobatae Ti thonia rotundifolia 4 1 Ti thonia diversifolia l OO 73 Tithonia koelzii Tithonia thurberi

100 ~~~~~~~~~~~~ser. 84t X < { = V. rhormbifolia Grammatoglossae 5 V. benziorum l00 Simsia villasenori Simsia ghiesbreghtii 1 Simsia sylvicola 69 Simsia annectens Simsia fruticulosa

1 v 73 Simsia lagascaeformis Simsia spooneri Simsia sanguinea 61 4 4 3 - Simsia foetida 84 Simsia amplexicaulis 2 V. sessilifolia 1 2 V. pringlei .53 83 V. seemannii V. purpusii ser. 3 Grammatoglossae V. grammatoglossa 53 V. bombycina V. davilae V. parkinsonii

V. linearis sect. Leighia 2 V. flava

83 2 V. pachycephala 89 V. excelsa V. hypochlora subg. V. ensifolia Amphilepis 2 V. angustifolia 1 6 V. buddleiformis 1 100 V. hemsleyana V. schultzii 1 Ti thonia peduncula ta 3 4 ~~~Tithonia cal va 99 - Ti thonia l ongiradiata Ti thonia tubaeformis

9 V. szyszyl owiczii sect . Paradosa 1 100 -- Simsia domnbeyana 944 AMERICAN JOURNAL OF BOTANY [Vol. 83

82 Arnica

30 F. monticola 15 H. multiflora 34 H. annuus

6 V. pinnatilobata33 1 V. pinnatilobata2502 8 7 P. lehmannii 2 10 V.flava 5 11 V. szyszylowiczii 1 9 4 T. rotundifolia 13 27 T. thurberii 3 T. longiradiata

9 T. calva

7 . calva

7 2 S. 3 dombeyana 193 1 S. dombeyana193b

9 5 - S. dombeyana306 4 I IIS. foetida

8 3 .2{1 V. ovata

1 V. rhombifolia 11 V.grammatoglossa 19 V. sessilifolia 11 V.puruana

21 V. tomentosa

Fig. 2. Strictconsensus tree of 12 most parsimonious cladograms based on sequence data fromthe nuclear ribosomal internaltranscribed spacer (ITS) region for samples of subtribeHelianthinae and outgroups. Decay index values shown above branches; bootstrapvalues shown below branches. sess the possibilitythat convergent morphology and ITS index to 0.64. A strictconsensus of cpDNA and ITS trees homoplasyhad occurred.A runthat placed the constraint for the entiredata set is given in Fig. 4. thatSimsia dombeyana and Viguiera szyszylowicziiform a monophyleticgroup (as in the cpDNA tree) produced DISCUSSION a shortesttree of 439 steps,an increase of 17 steps (4%). A second run with the constraintthat Tithonia rotundi- Overall phylogenyof Helianthinae based on cpDNA folia, T. thurberi,Simsia calva, and S. foetida be mono- restrictionsite data-The resultsreported here (Table 2; phyleticproduced a shortesttree of 441 steps,an increase Fig. 1) were mostlyconsistent with previous surveysof of 19 steps (4%). cpDNA restrictionsite variationin subtribeHelianthinae Combination of cpDNA and ITS data-Reanalysis of (Schilling and Jansen,1989; Schilling and Panero, 1991; the cpDNA restrictionsite data fromthe same samples Schilling, Panero, and Eliasson, 1994), but extended used for ITS sequence analysis (no data were available theseby providingnew or additionalinformation for Sim- forArnica mollis) resultedin a single fullyresolved tree sia, Tithonia, Viguiera ser. Grammatoglossae, and V. (Fig. 4), witha lengthof 59 steps and a consistencyindex subg. Amphilepis.Previous studies suggest thatthe sub- of 0.90. Combinationof cpDNA and ITS data sets yield- tribecan be divided into several basally divergentgroups ed a single tree with a lengthof 500 steps and a consis- (V. subg. Bahiopsis; Heliomeris; V. sect. Diplostichis), tency index of 0.58. Removal of all samples of S. dom- two groupsthat split off successively (V. sect. Maculatae beyana and of Tithonia,however, raised the consistency and Helianthus), and a pair of clades that include the July 1996] SCHILLING AND PANERO-PHYLOGENETIC RETICULATION IN HELIANTHINAE 945

Arnica F. monticola H. multiflora H. annuus >3 I V. pinnatilobata33 99 /I V. pinnatilobata2502 P. lehmannii

>3 3 V.flava 84% 87% V. szyszylowiczii >3 T. rotundifolia 92% >3 T. thurberi 97% 2 3= T. longiradiata 78% T. calva

S. calva

2 1 A = S. dombeyana193 49% 48% >3 S. dombeyana193b 96%/c89% S. dombeyana306 S. foetida

28% 3 | V. ovata 2 99% V. rhombifolia 55% V. grammatoglossa V. sessilifolia V. tomentosa V. puruana

Fig. 3. One of 12 most parsimoniouscladograms based on sequence data fromthe nuclear ribosomal internaltranscribed spacer (ITS) region, showingrelative branch lengths for samples of subtribeHelianthinae and outgroups.

remainingspecies. All of thenewly reported species were nificantlyin two places. One was in the placementof S. placed by cpDNA data into one or the other of latter dombeyana relative to othermembers of Simsia and to clades. South American Viguiera. The second was in the place- ment of members of Tithonia. The incongruentresults Overall phylogenyof Helianthinae based on ITS se- were clearly documentedby the collapse of all terminal quence data-The estimateof overall phylogenyof He- nodes in the strictconsensus of the most parsimonious lianthinaebased on ITS sequence data (Figs. 2, 3) mostly cpDNA- and ITS-based trees(Fig. 4). The relativelylarge was concordantwith thatbased on cpDNA (Figs. 1, 4). increases in tree lengthsobserved with constraintanaly- Both sets of data placed Heliomeris and Viguiera subg. ses provided furthersupport for this result by showing Bahiopsis in relativelybasal positions. The positions of that a large amount of homoplasy would be needed to V. sect. Maculatae and Helianthus as the nextrespective bringthe cpDNA and ITS data sets into accord withone groups to divergewithin Helianthinae were reflectedex- another.In both cases, the ITS-based tree appears to be actlyin thetwo analyses. Finally,the presence of samples concordantwith morphologicalobservations. Thus, ex- of Pappobolus, Simsia, Tithonia,V. ser. Pinnatilobatae, planationsbased on chloroplasttransfer are suggestedfor V. ser. Grammatoglossae,V. subg. Amphilepis,and the both S. dombeyanaand Tithonia.Lineage sorting(Doyle, South Americanspecies of Viguiera as relativelyderived 1992) is anotherpossible explanation,but it seems very species were all shown in both trees. The tree based on unlikelybecause it would requirean ancestralpopulation ITS sequence data did not, however,show clearly a di- with a degree of divergence between differentcpDNA vision of the relativelyderived species into two separate phenotypesfar greaterthan has been observed in any clades. In addition,there were significantdifferences be- extantspecies of Helianthinae. tweenthe cpDNA and ITS treesin theplacement of some individual species in the relativelyderived groups. Simsia dombeyana-The occurrence of a discordant cpDNA phenotype within Simsia was unexpected be- Incongruence between results based on cpDNA and cause the genus, includingS. dombeyana,appears to be ITS data-The cpDNA- and ITS-based treesdiffered sig- well characterizedmorphologically (Spooner, 1990). Sim- 946 AMERICAN JOURNAL OF BOTANY [Vol. 83

Flourensia monticola

10 15 V. tomentosa

Heliomeris multiflora

2 13 Viguiera puruana

Helianthus annuus

4 Pappobolus lehmanii 7 1 V. pinnatilobata 5 5 1 Simsia calva 1 ~~~9 1 Simsia foetida

2 V. ovata 7 V. rhombifolia 2 3 4 Tithonia thurberi

1 Simsia dombeyana 193

Simsia dombeyana 306

V. szyszylowiczii

V. grammatoglossa

2 V. sessilifolia

V. flava

4 Tithonia longiradiata

Tithonia calva

Fig. 4. Single most parsimoniouscladogram based on chloroplastDNA restrictionsite data (leftside of figure)and strictconsensus tree (right side of figure)combining the 12 most parsimoniouscladograms based on sequence data fromthe nuclearribosomal DNA internaltranscribed spacer (ITS) region (see Fig. 3) with the single most parsimoniouscladogram based on chloroplastDNA restrictionsite data. Numbers of site changes shown above branches. sia exhibitsa singular(within Helianthinae) and striking notype of S. dombeyana is that its cpDNA genome is morphological apomorphy in its strongly flattened derived from a chloroplastcapture event followinghy- achenes as well as several otherdistinctive traits (Spoon- bridizationwith a South American memberof Viguiera. er, 1990). All of the sampled species of the genus, except The South Americanmembers of Viguieraappear to form one, furthershared a characteristicsuite of cpDNA re- a single, relativelyderived, monophyletic lineage based strictionsite features(Fig. 1). The exceptionalspecies, S. on cpDNA data (Schilling, Panero, and Eliasson, 1994). dombeyana,is one of only two South Americanendemics ITS sequence resultsadd furtherweight to this explana- in this primarilyMexican genus (the otherSouth Amer- tion by showing that,based on this nuclear gene region, ican endemic,S. fruticulosa,exhibited a cpDNA pheno- S. dombeyana was the sistergroup to and differedlittle typesimilar to thatof otherSimsia species), and it occurs fromS. foetida (Fig. 2). Otherthan its distinctivecpDNA in highland areas in Ecuador and Peru, and Ar- genome, there was no clue that S. dombeyana had ac- gentina,and in easternBrazil (Spooner, 1990). Morpho- quired any othertraits from South Americanmembers of logical and flavonoiddata both suggest thatS. dombey- Viguiera, althoughdetailed and comprehensiveanalyses ana is not only accuratelyplaced in Simsia but is a near of morphologicalor chemical traitshave not been made. relativeto one of the most distinctivespecies, S. foetida (Schilling and Spooner, 1988; Spooner, 1990). In con- Tithonia-Available informationfrom cpDNA and trast,cpDNA data fromfour different samples of S. dom- ITS sequence studies suggested that phylogeneticretic- beyana (two each from Ecuador and Argentina) were ulation is also presentin the historyof Tithonia,where strikinglydifferent from all otherSimsia species but were it may be of even greaterinterest than in S. dombeyana. identicalto South Americanmembers of Viguiera.Based Morphological and phytochemicalstudies indicate that on morphologyand currentgeographic distributions, the Tithonia is distinctive and probably monophyletic most likely explanationfor the discordantcpDNA phe- (LaDuke, 1982a, b). An initialsurvey gave the surprising July 1996] SCHILLING AND PANERO-PHYLOGENETIC RETICULATION IN HELIANTHINAE 947 resultthat the single species of Tithoniaanalyzed, T. ro- by cpDNA or ITS sequence data, except for V. sessili- tundifolia,has a cpDNA restrictionsite patternalmost folia. This agreed with previous observationsbased on identical to those of samples of Simsia (Schilling and morphology(Panero and Schilling, 1992) that suggest Jansen,1989). A close relationshipbetween the two gen- thatSimsia should be enlargedto include these additional era has never been suggested. The additional survey of species. A second group consists of shrubbyspecies (V. cpDNA data reportedhere would suggest that Tithonia bombycina,V. davilae, V. grammatoglossa,and V. pur- is polyphyletic,with its species exhibitingtwo distinctly pusii; no data are available for V. hidalgoana, another differentcpDNA phenotypes(Fig. 1). The divisionof the apparentmember of the group), which are definedby a genus by cpDNA phenotypedoes not correspondto the morphologicalapomorphy in the patternof the basal fo- infragenericclassification suggested by LaDuke (1982c) liar veins (Schilling and Panero, 1990), and these were or to any simple combinationof morphologicalor phy- suggestedby cpDNA data to forma monophyleticgroup. tochemical features. Results of ITS sequence studies, A thirdgroup (V. pringleiand V. seemannii)also consists however,agreed with morphologyin suggestingthat Ti- of woody species, and cpDNA data suggestedthat this is thoniais monophyletic,and thusindicated the possibility a monophyleticgroup. The fourthgroup is formedby the thatone of the cpDNA phenotypeswithin the genus was single,morphologically distinctive, species, V. parkinson- acquired throughchloroplast capture. ii. The cpDNA data placed it in a clade with several Two facets of the informationregarding Tithonia groups, including V. subg. Amphilepis with which it would suggestthat the cpDNA transferis not recent.One shares similartraits of habit and geographicdistribution. is that several relativelydistinctive species of Tithonia The remainingpuzzle in V. ser. Grammatoglossaein- sharedthe cpDNA phenotypeof the Simsia lineage. This volves the relationshipsof V. sessilifolia. Morphologi- would suggest that diversificationto produce these spe- cally this species clearly resembles Simsia in habit and cies occurredafter the cpDNA transfer.The second is that in featuresof the synflorescence,involucre, and floralmi- the samples of Tithoniaplaced near Simsia differedfrom cromorphology.Neither cpDNA nor ITS sequence data all Simsia samples by lack of an apomorphicsite change. suggestthat it is partof the Simsia clade, however(Figs. Althoughit is possible that a reversionhas occurredin 1, 2). The morphological similaritieswith Simsia may the Tithoniaspecies, the more parsimoniousexplanation thus simplyreflect homoplasious changes. It is also pos- is thatthe cpDNA transferoccurred prior to divergence sible, however,that the morphologicalfeatures provide withinSimsia. The presence of evidence for a cpDNA evidence of a phylogeneticrelationship to Simsia thatis transferthat is not relativelyrecent is excitingand sug- not reflectedin either cpDNA or ITS sequence data. gests thatTithonia forms an example of phylogeneticre- Analysis of other,nuclear DNA markersin this species ticulationthat may be of exceptional interestfor the as- may be necessary to resolve its phylogeneticplacement. sessmentof the evolutionaryrole of hybridization. A weakness of the hypothesisof chloroplasttransfer LITERATURE CITED to explain the incongruencebetween cpDNA and ITS trees is that hybridsfrom wide crosses in Helianthinae BALDWIN, B. G. 1992. Phylogeneticutility of the internaltranscribed spacers of nuclear ribosomalDNA in : an example fromthe are not known fromnature. They have, however,been Compositae. Molecular Phylogeneticsand Evolution 1: 3-16. reportedfrom artificial crosses. Some Tithoniaand Sim- . 1993. Molecular phylogeneticsof Calycadenia (Compositae) sia species occupy similar weedy, roadside habitatsin based on its sequences of nuclear ribosomal DNA: chromosomal Mexico, where the presence of numerous individuals and morphologicalevolution reexamined. American Journal of Bot- growingin close proximitywould produce abundantop- any 80: 222-238. portunitiesfor crossing. It is not certain that a natural BLAKE, S. F 1918. A revisionof the genus Viguiera. Contributionsof hybridwould be noted or recognizedas such in thishab- the Gray Herbarium54: 1-205. BREMER, K. 1994. : cladistics and classification.Timber itat. Artificialhybrids between T. rotundifoliaand He- Press, Portland,OR. lianthus annuus have been reported from sunflower CRISTOV, M., AND I. PANAYOTOV. 1991. Hybrids between the genera breeding programs(Cristov and Panayotov, 1991). The Helianthus and Tithoniaand theirstudy. Helia 14: 27-34. potentialfor wide hybridizationmay be enhanced in He- DONOGHUE, M. J., R. G. OLMSTEAD, J. F SMITH, AND J. D. PALMER. lianthinaeby the outcrossingbreeding system and open, 1992. Phylogeneticrelationships of Dipsacales based on rbcL se- of floral characteristicof the quences. Annals of the Missouri Botanical Garden 79: 333-345. unspecialized type display DOYLE, J. J. 1992. Gene treesand species trees:molecular systematics subtribe. as one-charactertaxonomy. Systematic Botany 17: 144-163. , AND J. L. DOYLE. 1987. A rapid DNA isolation procedurefor Relationships of species of Viguiera ser. Grammato- small quantitiesof freshleaf tissue.Phytochemical Bulletin 19: 11- glossae-Molecular data (Figs. 1, 2) supportedprevious 15. suggestionsthat V. ser. Grammatoglossaeas definedby FELSENSTEIN, J. 1985. Confidencelimits on phylogenies:an approach Blake (1918) is a heterogeneousgroup and Pa- using the bootstrap.Evolution 39: 783-791. (Schilling GRANT, V. 1981. Plant speciation,2d. ed. Columbia UniversityPress, nero, 1990; Spooner, 1990; Panero and Schilling, 1991), New York, NY. and provided hypothesesfor the circumscriptionand re- JANSEN,R. K., AND J. D. PALMER. 1987. ChloroplastDNA fromlettuce lationshipsof its componentspecies groups. Species of and Barnadesia (Asteraceae): structure,gene localization,and char- V. ser. Grammatoglossaecan be divided into fourgroups acterizationof a large inversion.Current Genetics 11: 553-564. based on morphology.One groupconsists of several spe- , AND . 1988. Phylogeneticimplications of chloroplast DNA restrictionsite variationin the Mutisieae (Asteraceae). Amer- cies (V. benziorum,V. ovata, V. rhombifolia,and V. ses- ican Journalof Botany 75: 753-766. silifolia) thatare primarilyherbaceous and share a num- LADUKE, J. C. 1982a. Revision of Tithonia.Rhodora 84: 453-522. ber of key morphologicaltraits with Simsia (Panero and 1982b. Flavonoid chemistryand systematics of Tithonia Schilling, 1991). Of these, all were placed with Simsia (Compositae). AmericanJournal of Botany 69: 784-792. 948 AMERICAN JOURNAL OF BOTANY [Vol. 83

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