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Home , Adoxa, Adoxaceae, Adoxa moschatellina, Caprifoliaceae, Centranthus, Centranthus ruber

PHYLOGENY AND PHYLOGENETIC OF , WITH SPECIAL REFERENCE TO AND ()

MICHAEL J. DONOGHUE,1 TORSTEN ERIKSSON,2 PATRICK A. REEVES,3 AND RICHARD G. OLMSTEAD 3

Abstract. To further clarify phylogenetic relationships within Dipsacales,we analyzed new and previously pub- lished rbcL sequences, alone and in combination with morphological data. We also examined relationships within Adoxaceae using rbcL and nuclear ribosomal internal transcribed spacer (ITS) sequences. We conclude from these analyses that Dipsacales comprise two major lineages:Adoxaceae and (sensu Judd et al.,1994), which both contain elements of traditional Caprifoliaceae.Within Adoxaceae, the following relation- ships are strongly supported: ( ( (Sinadoxa (Tetradoxa, )))). Combined analyses of C ap ri foliaceae yield the fo l l ow i n g : ( C ap ri folieae (Diervilleae (Linnaeeae (Morinaceae ( (,)))))). On the basis of these results we provide phylogenetic definitions for the names of several major clades. Within Adoxaceae, Adoxina refers to the clade including Sinadoxa, Tetradoxa, and Adoxa.This lineage is marked by herbaceous habit, reduction in the number of perianth parts,nectaries of mul- ticellular hairs on the perianth,and bifid . The clade including Morinaceae,Valerianaceae, Triplostegia, and Dipsacaceae is here named Valerina. Probable synapomorphies include herbaceousness,presence of an epi- calyx (lost or modified in Valerianaceae), reduced endosperm,and distinctive chemistry, including production of monoterpenoids. The clade containing Valerina plus Linnaeeae we name Linnina. This lineage is distinguished by reduction to four (or fewer) stamens, by abortion of two of the three carpels,and possibly by supernumerary . Keywords: Adoxaceae, Caprifoliaceae, Dipsacales, ITS, morphological characters, phylogeny, phylogenetic taxonomy, phylogenetic nomenclature, rbcL, Sinadoxa, Tetradoxa.

Several studies of Dipsacales phylogeny have port for these clades has not been uniformly appeared in recent years, based on both mor- strong, and the exact placement of several key phological and molecular evidence (Donoghue, taxa (H ep t a c o d i u m, S i n a d ox a, Te t ra d ox a, 1983; Donoghue et al., 1992; Judd et al., 1994; Triplostegia) remains uncertain. Backlund and Donoghue, 1996; Backlund and Here we present an expanded analysis of Bremer, 1997; Pyck et al., 1999; Pyck and ch l o roplast DNA r b cL sequences fo r Smets, 2000; Olmstead et al., 2000). These Dipsacales, and a combined analysis of rbcL have concluded that Viburnum is related to with morphological characters. We focus spe- Sambucus plus Adoxa and that the remainder of cial attention on phy l ogenetic re l at i o n s h i p s the traditional Caprifoliaceae are more closely within Adoxaceae, based on a combination of re l ated to Mori n a c e a e,Va l e ri a n a c e a e, a n d nuclear ribosomal internal transcribed spacer Dipsacaceae. Furthermore, these studies have (ITS) and rbcL sequences. Of special interest is s u p p o rted the monophy ly of C ap ri fo l i e a e, the placement of the two rare and morphologi- Diervilleae, and Linnaeeae (all of traditional cally bizarre Chinese , Sinadoxa cory - Caprifoliaceae), and the view that Linnaeeae dalifolia and Tetradoxa omeiensis.The recent a re more cl o s e ly re l ated to Mori n a c e a e, rediscovery of these species allows us to pre- Valerianaceae, and Dipsacaceae. However, sup- sent molecular evidence on their relationships.

We are grateful to Jim Smith for rbcL sequencing efforts early in the life of this project, and to Bruce Baldwin for help with ITS sequencing.We also thank D. Boufford, B. Tan, K. In (Chengdu, Sichuan), T.-S.Ying (Beijing), and T.-N. Ho (Xining, Qinghai) for organizing expeditions to re-collect Tetradoxa and Sinadoxa, and for their help in the field.The expedition to Qinghai was supported by a National Geographic Society award to D. Boufford. Nancy Pyck and Chuck Bell shared information on their ndhF studies of Dipsacales, and Anders Backlund provided useful comments early on. 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, , 06511-8160 U.S.A. E-mail: [email protected] 2 Bergius Foundation, Royal Swedish Academy of Sciences, Box 50017, S-104 05 Stockholm, ,and Department of , Stockholm University, S-106 91 Stockholm, Sweden. 3 Department of Botany, Box 355325, University of , Seattle,Washington 98195-5325, U.S.A.

Harvard Papers in Botany, Vol. 6,No. 2, 2001, pp. 459Ð479. © President and Fellows of Harvard College, 2001. 460 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

On the basis of these findings we contrast a strands. ITS sequences were edited using the p hy l ogenetic nomencl at u ral system fo r Staden package (Staden, 1996) under the Linux Dipsacales with a traditional Linnaean taxo- operating system. nomic treatment, and present phylogenetic def- initions for the names of several major clades. Alignment rbcL nucleotide sequence data were aligned MATERIALS AND METHODS manually using the editor in PAUP* (Swofford, Taxa 2001); no indels were recorded.The rbcL data Our rbcL analyses included 30 species of tra- matrix contained 2% “missing data” for the 43 ditional Dipsacales, covering all major lineages t a x a , re flecting uncertainty in base-calling. (Table 1). We also included Columellia and Primer positions 1Ð26 were excluded from the , which may be closely related to, analyses. Of the remaining 1402 characters, or possibly even nested within, D i p s a c a l e s 230 were parsimony informative. (Backlund and Donoghue, 1996; Backlund and ITS nucleotide sequences from 15 taxa were Bremer, 1997). Previous analyses have shown aligned manually using the Se-Al alignment t h at Dipsacales belong within A s t e ri d a e editor (Rambaut, 1 9 9 6 ) , resulting in 679 (Olmstead et al., 1992, 1993; Chase et al., aligned positions. Positions 414Ð482 we re 1993; Backlund and Bremer 1997), where they removed from parsimony analyses owing to are related to and within a difficulties in alignment. Indels were treated as “euasterid II” clade (APG, 1998). Here we have “uncertain” in the matrix, and 21 parsimony- included published rbcL sequences from four informative indels (positions 47, 48, 53, 92, species of Apiales (Coriandrum, Griselinia, 111Ð112, 129, 166, 203, 210Ð212, 216, 221, H e d e ra, P i t t o s p o ru m) , and four species of 222, 235, 372, 375, 405, 572, 581, 607Ð608, A s t e rales (B a rn a d e s i a, B o o p i s, C a m p a nu l a, 6 0 9 , and 631) we re coded as binary Me n yan t h e s ). In addition, we added Gentiana presence/absence characters and added to the and Nicotiana as representatives of the “euas- end of the matrix. Our ITS matrix contained terid I” clade (APG, 1998), and Cornus of 6.2% uncertain data: 0.4% due to uncertain . On the basis of all previous phyloge- scorings, 2.8% to implied indels, and 3.0% to netic analyses, our were rooted along the filling in unsequenced portions of the 5.8S gene branch connecting Cornus. in several species. In total, our ITS matrix con- Our ITS analyses included new sequences tained 181 parsimony-informative characters. fr om Tet ra d ox a and Si n a d ox a (T able 1), al o n g with five sequences from Sa m bu c u s (E ri k s s o n Combining Data and Donogh u e , 1997) and five from Vibu r num We analyzed five datasets. Three of these (D o n o ghue and Baldwi n , 1993; Baldwin et al., focused on Dipsacales: (1) rbcL alone for 1995; see also Eriksson and Donogh u e , 19 9 7 ) . Dipsacales and outgroups, (2) rbcL plus mor- Sequences of Di e rv i l l a and Wei ge l a (D i e rv i l l e a e ) , phological data from Judd et al. (1994), and (3) obtained from Genbank (Kim and Kim, 1999), rbcL plus morphological data from Backlund were included for rooting purposes. and Donoghue (1996). All data from the mor- phological studies were added without changes Sequencing in the characters or in taxon scoring; both Of the 43 rbcL sequences included in our datasets also included chromosome and sec- analyses, 27 were obtained from the authors of ondary chemical characters. Two additional previous studies or from Genbank (Table 1) and datasets focused on Adoxaceae: (4) ITS alone 16 are reported here for the first time.The new for Adoxaceae and outgroups, and (5) ITS plus sequences were obtained using standard proto- rbcL. Here we describe assembly of the com- cols and primers (see, e.g., Olmstead et al., bined datasets (2, 3, and 5). 1992, 1993). ITS sequences for Sinadoxa and The Judd et al. (1994) morphological analy- Te t ra d ox a we re obtained using pro t o c o l s sis included a subset of the taxa in our rbcL described elsewhere (Eriksson and Donoghue, matrix. From our rbcL dataset we omitted 12 1997); however, we used a different 5' primer outgroup taxa not present in Judd et al., keep- (ITS-I of Urbatsch et al., 2000), and reads were ing only Pittosporum for rooting purposes. long enough that internal primers were not Judd et al. included Alseuosmiaceae, but these n e e d e d. All sequences rep o rted here we re are probably not closely related to Dipsacales p ro o f read using sequences of both DNA (Gustafsson et al., 1996) and have been omitted 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 461 here. We also omitted rbcL sequences for those were possible for datasets 2, 4, and 5. All char- Dipsacales not rep resented in Ju dd et al.: acters were weighted equally, and indels were , Morina, Sinadoxa, Tetradoxa, t re ated as described ab ove. MacClade and and Triplostegia. Judd et al. lumped PAUP* were used for the output of trees, char- and together, and also Kolkwitzia and acter optimizations, and so on. , because these scored identically for their 35 (25 binary, 10 multistate) morphologi- Measures of Support cal characters. In our analysis each of these Decay indices (Bremer, 1988; Donoghue et pairs was separated into two taxa, which were al., 1992) were calculated using AutoDecay scored identically for all morphological charac- (Eriksson, 1999) and PAUP*. The converse ters. Several taxa in Judd et al. were repre- constraint runs in PAUP* were performed using sented by two or more rbcL sequences in our branch-and-bound searches when possible; oth- matrix. Using the “merge taxa” function in erwise, heuristic searches were conducted with MacClade (Maddison and Maddison, 1992), 100 random addition sequence starting trees, we merged the sequences of our two Sambucus TBR branch swapping, and saving all most par- species into a single sequence with six poly- simonious trees. Bootstrap analy s e s m o rphic positions. Similarly, we merge d (Felsenstein, 1985) were carried out using 500 sequences of Vi bu rnu m ( t h ree species), rep l i c ations with simple addition sequence, L o n i c e ra ( t wo species), Dipsacaceae (thre e TBR branch swapping, and saving all most par- s p e c i e s ) , and Va l e rianaceae (five species), simonious trees in each replicate. resulting in 28, 55, 40, and 100 polymorphic positions, respectively. The resulting combined RESULTS matrix contained 16 taxa and 1437 characters Pa rs i m o ny analysis of our r b cL mat ri x (after excluding primer sites 1Ð26); there were (analysis 1) resulted in four minimum-length 93 parsimony-informative characters. trees of 996 steps (CI = 0.521; CI excluding The Backlund and Donoghue (1996) dataset uninformative = 0.404; RI = 0.593); these dif- contained many more taxa (58) and characters fer only in whether S y m p h o ri c a rp o s o r (109). However, owing to differences in taxon is more closely related to Lonicera sampling, we removed 9 outgroup taxa from (Fig. 1). This result supports many previous our r b cL dataset (B a rn a d e s i a, B o o p i s, conclusions. Dipsacales are seen to be mono- Campanula, Coriandrum, Cornus, Gentiana, phyletic with Columellia and Desfontainia as H e d e ra, M e nya n t h e s, and N i c o t i a n a) . their sister group. This is in contrast to several Likewise, from the morphological dataset we p revious rep o rts that C o l u m e l l i a a n d removed 12 Dipsacales and 16 outgroup taxa. Desfontainia may be nested within the group Sambucus, Viburnum, and Lonicera sequences (e.g., Backlund and Donoghue, 1996; Backlund were merged as described above. The com- and Bremer, 1997; see discussion). We find lit- bined dataset contained 30 taxa and 1511 char- tle support for a connection betwe e n acters (after excluding primer sites 1Ð26); there A d oxaceae and A raliales (Backlund and were 236 parsimony-informative characters. Bremer, 1997); forcing these taxa together adds The combined analysis of Ad o xaceae used the a minimum of five steps. The tra d i t i o n a l i n t e rsection of all ava i l able ITS and r b cL Dipsacaceae and Valerianaceae are both mono- sequences. It contained 10 taxa and 2012 cha ra c - phyletic, show internal relationships largely te r s (after exc luding primer sites 1Ð26 and 69 ITS consistent with previous results (but see Caputo sites in a region of ambiguous alignment); there and Cozzollino, 1994, on Dipsacaceae), and are wer e 229 parsi m o ny - i n fo rm at i ve cha ra c t e rs . weakly united with Triplostegia. In our rbcL All of our data mat ri c e s , and the tre e s analysis, it is equally parsimonious to place rep o rted here, a re ava i l able in Tre e BA S E Triplostegia as the sister group of Valerianaceae (http://treebase.org). (as in previous studies: B a cklund and Phylogenetic Analyses Donoghue, 1996; Backlund and Bremer, 1997; Parsimony analyses were conducted using see discussion) or Dipsacaceae. M o ri n a PAUP* (Swofford, 2001). For datasets 1 and 3 ( M o rinaceae) is sister to the Dipsacaceae- (see above), we carried out heuristic searches Triplostegia-Valerianaceae clade. (1000 random addition sequence starting trees, Caprifoliaceae, in the traditional sense, are TBR branch swapping, saving all most parsi- cl e a rly not monophy l e t i c. Vi bu rnu m a n d monious trees); bra n ch-and-bound search e s Sambucus are united with Adoxa and its rela- 462 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

FIG U R E 1. Strict consensus of four most parsimonious trees resulting from the analysis of rbcL sequence data alone (analysis 1); length=996 steps; consistency index (CI), exc luding uninform at i ve cha ra c t e r s=0.40; ret e n t i o n in d e x (RI)= 0.59. Bootstrap values grea ter than 50% are indicated abo ve the bran c hes and decay indices below. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 463 tives, with Sinadoxa seen to be the sister group Valerianaceae, Triplostegia, and Dipsacaceae. of A d ox a plus Te t ra d ox a. Cap ri fo l i e a e, The combined analysis also united Triplostegia Diervilleae, and Linnaeeae, of the traditional with Va l e ri a n a c e a e, instead of with Caprifoliaceae, are more closely related to the Dipsacaceae as in the rbcL analysis. M o ri n a c e a e - D i p s a c a c e a e -Tri p l o s t egi a- Our analysis of Adoxaceae based on ITS Valerianaceae clade. Caprifolieae are mono- sequences alone (analysis 4) resulted in two phyletic, with the sister group of a trees of 347 steps (Fig. 5; CI = 0.784; CI clade including S y m p h o ri c a rp o s, Tri o s t e u m, excluding uninformative = 0.750; RI = 0.864). and Lonicera.This clade is in turn weakly As in previous analyses (Donog h u e, 1 9 8 3 ; united with H ep t a c o d i u m. Dierv i l l e a e D o n oghue et al., 1992; Ju dd et al., 1 9 9 4 ; (D i e rv i l l a, Wei ge l a ) are also monophy l e t i c. B a cklund and Donog h u e, 1 9 9 6 ) , Vibu r nu m However, in contrast with previous analyses, appears to be the sister group of a clade con- Linnaeeae do not form a clade in this analysis. taining Sambucus and Adoxa.The significant is separated from Abelia, Dipelta, and new result is strong support for the placement Kolkwitzia, which are in turn paraphyletic in of S i n a d ox a and Te t ra d ox a in re l ation to relation to Diervilleae. Branch lengths in this Adoxa. As in the rbcL analyses just discussed, portion of the are especially short (Fig. 2), Sinadoxa is the sister group of a Tetradoxa- i m p lying that other arra n gements may be Adoxa clade (also see Backlund and Donoghue, nearly as parsimonious. Trees constrained to 1996). As expected, the combined analysis of unite Abelia, Dipelta, and Kolkwitzia are only ITS and rbcL sequences for Adoxaceae (analy- two steps longer than our shortest trees; how- sis 5) yielded a single tree of 395 steps (CI = ever, linking Linnaea to this group, in a mono- 0.858; CI excluding uninformative = 0.828; RI phyletic Linnaeeae, yields trees that are five = 0.881) with remarkably high support values steps longer. for all clades (Fig. 6). Parsimony analyses of rbcL sequences com- bined with morphological characters yield sev- DISCUSSION eral important insights. Combining rbcL with Phylogenetic Relationships the Judd et al. (1994) morphological dataset With the availability of one or more rbcL (analysis 2) yielded five trees of 258 steps (Fig. sequences for all of the standardly recognized 3; CI = 0.767; CI excluding uninformative = ge n e ra of the traditional Cap ri fo l i a c e a e 0.657; RI = 0.757). These trees are entirely (, a segregate of the traditional Abelia, congruent with those obtained by Judd et al. is not included here), and a reasonable sample (1994) using morphology alone, and in almost of Va l e rianaceae and Dipsacaceae, m a ny every respect they are also congruent with the aspects of the phylogeny of Dipsacales are now rbcL trees discussed above. However, in con- established with considerable certainty. Most t rast to results from analyses using r b cL i m p o rt a n t ly, it is cl e a r, as highlighted by sequences alone, the Linnaeeae form a weakly Donoghue (1983), that the traditional circum- s u p p o rted cl a d e, wh i ch is sep a rated fro m scription of Caprifoliaceae must be abandoned Diervilleae and instead united directly with (also see Donoghue et al., 1992; Judd et al., Dipsacaceae and Valerianaceae. 1994; Backlund and Donoghue, 1996; and later Combining r b cL with the Backlund and authors). Viburnum and Sambucus are more D o n oghue (1996) morp h o l ogical ch a ra c t e rs closely related to Adoxa and its relatives than (analysis 3) yielded 12 trees of 861 steps (Fig. they are to the other traditional Caprifoliaceae. 4; CI = 0.569; CI excluding uninformative = Caprifolieae, Diervilleae, and Linnaeeae, of the 0.478; RI = 0.689). Almost all of the clades in t raditional Cap ri fo l i a c e a e, a re more cl o s e ly these trees correspond with those found using re l ated to Mori n a c e a e,Va l e ri a n a c e a e, m o rp h o l ogical ch a ra c t e rs alone, r b cL Triplostegia, and Dipsacaceae than they are to sequences alone, and rbcL plus the Judd et al. Vibu r nu m and Sa m bu c u s . Of these gro u p s , morphological characters. The main difference Linnaeeae appear to be more closely related to is between this combined analysis and the rbcL the Mori n a c e a e - Va l e ri a n a c e a e -Tri p l o s t egi a- sequences analyzed separately. Although sup- Dipsacaceae clade than to Cap ri folieae or port values are not high, here again the com- Diervilleae. bined data yield a monophyletic Linnaeeae, For purposes of the remaining discussion we which is separated from Diervilleae and united will adopt the names for the two major clades of with a clade including Mori n a c e a e, Dipsacales suggested by Jud d et al. (1994). 464 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

FIG U R E 2. Phylogram from the rbcL analysis; branch lengths scaled to the number of inferred state changes. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 465

FIG U R E 3. Strict consensus of five most parsimonious trees resulting from the analysis of rbcL sequence data combined with the morphological characters from Judd et al. (1994) (analysis 2); length=258 steps; consis- tency index (CI), excluding uninformative characters=0.66; retention index (RI)= 0.76. Bootstrap values greater than 50% are indicated above the branches and decay indices below. 466 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

FIG U R E 4. Strict consensus of 12 most parsimonious trees resulting from the analysis of rbcL sequence data combined with the morphological characters from Backlund and Donoghue (1996) (analysis 3); length=861 steps; consistency index (CI), excluding uninformative characters=0.48; retention index (RI)=0.69. Bootstrap values greater than 50% are indicated above the branches and decay indices below. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 467

FIG U R E 5. Phylogram of one of the two most parsimonious trees resulting from the analysis of Adoxaceae based on ITS sequences (analysis 4); length=347 steps; consistency index (CI), excluding uninformative char- acters=0.75; retention index (RI)=0.86. Bootstrap values greater than 50% are indicated above the branches and decay indices below. Branch lengths scaled to the number of inferred state changes. 468 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

FIG U R E 6. The single most parsimonious trees resulting from the analysis of Adoxaceae based on rbcL and ITS sequences (analysis 5); length=395 steps; consistency index (CI), excluding uninformative characters=0.83; retention index (RI)=0.88. Bootstrap values greater than 50% are indicated above the branches and decay indices below. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 469

Ad o xaceae will ref er to the clade inclu d i n g them wer e unres o l ve d . All of our molecular Vibu r num , Sa m bu c u s , Si n a d ox a , Tet ra d ox a , an d an a ly s e s , based on rb c L alone, ITS alone, an d Ad ox a . Cap ri foliaceae will ref er to the cla d e the two combined, s u p p o rt the S i n a d ox a- i n cluding Cap ri fo l i e a e, D i e rv i l l e a e, a n d Tet ra d ox a -Ad ox a cla d e , with bootstrap values of Linnaeeae (of traditional Capri fo l i a c e a e ) , pl u s 100% in each case, and decay values of 10 for Mo ri n a c e a e ,Val e ri a n a c e a e , Trip l o s t e gia , an d rb c L, 34 for ITS, and 42 for the combined data . Di p s a c a c e a e . Note especially that Capri fo l i a c e a e These analyses also support the conclusion that se n s u Jud d et al. (1994) includes sever al taxa Ad ox a and Tet ra d ox a ar e more clo s e l y rel a ted to t ra d i t i o n a l ly tre ated as families (e. g. , one another than either is to Si n a d ox a . Bootstrap Di p s a c a c e a e ,Val e ri a n a c e a e ) , whose names we and decay values are high for the Tet ra d ox a - retain here (see below under “ P hy l ogen e t i c Ad ox a cla d e : 89% and decay = 2 for rb c L alone, Tax o n o m y”). Ad ox a c e a e , Cap ri fo l i a c e a e , an d 100% and decay = 8 for ITS alone, and 100% the names of other major clades are given form a l and decay = 9 for the combined analysis. phy l o genetic definitions below. These results bear importantly on our inter- pretation of character evolution in Adoxaceae. Relationships and evolution in Adoxaceae. The fi rst split, b e t ween Vibu r nu m and the Relationships within Adoxaceae now seem S a m bu c u s-A d ox a clade (Adoxoideae s e n s u firmly established. Sambucus and Adoxa have Thorne, 1992; Eriksson and Donoghue, 1997) previously been united on the basis of morpho- is marked by several morphological changes. logical characters (Donoghue, 1983; Judd et Abortion of two of the three carpels and dis- a l . , 1994; Backlund and Donog h u e, 1 9 9 6 ) , placement of the remaining fertile ovule into a rbcL sequences (e.g., Donoghue et al., 1992; s t e rile carpel (Wi l k i n s o n , 1 9 4 8 , 1 9 4 9 ; Chase et al., 1993; Backlund and Bre m e r, Fukuoka, 1972) presumably evolved along the 1 9 9 7 ) , and ITS sequences (Eriksson and Viburnum branch (Donoghue, 1983); the result- D o n og h u e, 1997). The re l ationships of ing one-seeded is also pro b ably a S i n a d ox a and Te t ra d ox a h ave, h oweve r, Viburnum synapomorphy. The Adoxoideae are received rather little attention, owing in large marked by the evolution of pinnately com- part to the rarity of these and, therefore, pound (or deeply dissected) , simple ves- the limited material available for study (but see sel perforations, extrorse anthers (at least at Liang, 1997). These taxa were both described maturity; see Erbar, 1994), vestigial archespo- from China in 1981—Sinadoxa by Wu et al. rial tissue (Erbar, 1994), Adoxa-type embryo (1981) from limestone outcrops near Nangqen sac deve l o p m e n t , and possibly by seve ra l in the southeast corner of Qinghai province, chemical characters, including production of and Tetradoxa by Hara (1981; Wu, 1981) from (Donoghue, 1983; Judd et al., 1994; Mt. Omei in Sichuan prov i n c e. Relocat i n g Backlund and Donoghue, 1996; Eriksson and these plants has proven difficult, however, and Donoghue, 1997). few additional specimens have come to light. In Within Adoxoideae, exactly which morpho- 1995 an expedition organized by Dr. In Kaipu logical changes mark the Sambucus branch of the Chengdu Botanical Institute was suc- remains uncertain, though candidate synapo- cessful in re d i s c ove ring Te t ra d ox a on Mt. morphies include crystal sand in cortical cells Omei. Later that summer, Sinadoxa was recol- and apical opening of the endocarp (see lected in southern Qinghai on an expedition Eriksson and Donoghue, 1997). In contrast, the j o i n t ly orga n i zed by the Xining Botanical S i n a d ox a-Te t ra d ox a-A d ox a clade is we l l Institute, the Beijing Botanical Institute, and marked by the evolution of the herbaceous the Arnold Arboretum of Harvard University. habit, with and a basal rosette of Both species appear to be extremely rare, but deeply lobed leaves; reduction of the perianth sufficient material was obtained to conduct the parts to four or fewer in at least some ; sequencing reported here (Table 1). nectaries of multicellular hairs positioned on Se ver al authors have argued that Ad ox a an d the perianth parts (see Wagenitz and Laing, Si n a d ox a ar e direc t l y rel at e d , with Tet ra d ox a 1984; Erbar, 1994); bifid stamens (arising from mo r e distant and retaining the largest number of a single primordium) in which the filaments are an c e s t r al feat u r es (Wu, 1981; Liang and Zhang, more or less deeply split and the monothecous 1986; Liang, 1997). These three taxa for med a anthers are therefore widely separated; separate clade in the morph o l o gical analysis of Backl u n d styles with small stigmas; and that are and Donoghue (1996), but rel a tionships among m o re - o r-less dry at mat u rity (possibly dis- 470 HARVARD PAPERS IN BOTANY Vol. 6, No. 2 persed by ants). These plants may also have a the five-merous flowers (also see Donoghue et distinctive chemistry, including loss of valeri- a l . , 1 9 9 8 ) , loss of early corolla ring pri- anic acid and the production of coumarins, mordium, and reduced vestigial archesporium. though this has not been studied in Sinadoxa and Tetradoxa. It is noteworthy that several of Relationships and evolution in Caprifoliaceae. these features appear to have evolved indepen- Comparison of our separate and combined dently within Sambucus, including herbaceous- analyses shows that, although these generally ness, reduction in the number of perianth parts, yield similar results, morphological characters and a tendency toward splitting of the stamens are important in resolving a number of rela- and styles (Eriksson and Donoghue, 1997). tionships. Perhaps most importantly, morpho- Within the Sinadoxa-Tetradoxa-Adoxa clade, l ogical data tend to unite the ge n e ra of Sinadoxa is unique by virtue of its spikelike Linnaeeae, greatly increase support for the structure (with scattered clusters M o ri n a c e a e - Va l e ri a n a c e a e -Tri p l o s t egi a- of three to five flowers), extreme variation and Dipsacaceae clade, and link these two clades reduction in the number of perianth parts, and d i re c t ly toge t h e r. Th ey also seem to link especially by the reduction to just a single Triplostegia with Valerianaceae, as opposed to carpel and the production of fruits with calyx- Dipsacaceae. d e rived carnose sacs (wh i ch may fa c i l i t at e Linnaeeae we re united by supernu m e ra ry water dispersal). The Tetradoxa-Adoxa clade is i n fl o rescence bracts (see Fukuoka, 1 9 6 9 ; characterized by smaller plants with leaves that Web e rl i n g, 1989) in Ju dd et al. (1994). are trifoliate or ternately lobed. In Tetradoxa, However, it is perhaps more likely that this fea- the perianth parts are acuminate at the apex (as ture is characteristic of Linnaeeae plus the they also are in several Sambucus species, M o ri n a c e a e - Va l e ri a n a c e a e -Tri p l o s t egi a- including S. ebulus; Eriksson and Donoghue, Dipsacaceae clade. Under this view, supernu- 1997); fusion of filament-halves at the base merary bracts were modified and fused to form (i.e., separation of filaments above the middle) the characteristic epicalyx seen in Morinaceae may also be an apomorphy of this species. The and Dipsacaceae (Hofmann and Göttmann, elongate inflorescence of Tetradoxa, with flow- 1990; Manchester and Donoghue, 1995; Roels ers on distinct pedicels, may represent the and Smets, 1996; Backlund and Donoghue, ancestral condition from which the headlike 1996). This scenario would, however, require inflorescence of Adoxa was derived by “com- the addition of a second ep i c a lyx in pression.” Although most Tetradoxa flowers are Tri p l o s t egi a and the loss of ep i c a lyx in 4-merous, they show considerable variation in Valerianaceae (or perhaps its modification to merousness along the inflorescence axis (Liang form wings, as in ). It is possible and Zhang, 1986); in Adoxa, the four lateral that Linnaeeae are distinguished by supervolute flowers in the cubelike head are typically five- vernation and by contraction of the chloro- merous (usually with only three devel- plast DNA inverted repeat, but data are still too oped), and the terminal is four-merous limited to be certain (Backlund and Donoghue, (see Erbar, 1994). 1996). The best support for the monophyly of Liang (1997) showed that Tet ra d ox a a n d Linnaeeae comes from analyses of ndhF and Sinadoxa have simpler floral vasculature than other chloroplast genes. Studies by Pyck et al. either or A. orientalis (1999), Pyck and Smets (2000), and Bell et al. (Nepomnyashchaya, 1984) and suggested that (2001) all support Linnaeeae as a clade. Tetradoxa, in particular, had retained the ances- Mo rp h o l o gical data provide more convi n c i n g tral characteristics (see also Liang and Zhang, s u p p o rt for the Mori n a c e a e - Va l e ri a n a c e a e - 1986). Our results suggest that elongate inflo- Trip l o s t e gia -Dipsacaceae cla d e , whi c h is only rescences may have been ancestral, but it is wea k l y supported in rb c L analyses. A num b e r p o s s i ble that the T- s h aped stamens of of cha ra c t e r s ref lect a shift to herbaceousness in Tetradoxa were derived from an ancestor with this grou p , in c luding the presence of a tapro o t the completely bifid condition, as seen in the and persistent basal leaves (in addition to other species. Inclusion of S i n a d ox a a n d cauline leaves ) , the absence of scales, and a Tetradoxa in developmental studies will be te n d e n c y towar d simple perfor ation plates in the necessary to determine the generality of many vessels (especially in Val e r ianaceae). Pos s i bl e other unusual characteristics seen in Adoxa repro d u c t i ve synapo m o r phies include the pres - (Erbar, 1994), such as “lobelioid” orientation of ence of an epi c a l yx (though, as noted abo ve, th i s 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 471 would req u i r e loss in Val e ri a n a c e a e ) , red u c t i o n Triplostegia has sometimes been linked with of the calyx lobes or modific a tion into persi s t e n t Valerianaceae and sometimes with Dipsacaceae br istles or pappus (more “n o rm a l ” lobes are seen in (see Backlund and Bremer, 1998). In an earlier Mo ri n a c e a e an d in Na rd o s t a c hys of Val e ri a n a c e a e), rbcL study (Backlund and Bremer, 1998) the and reduction in endosperm, cu l m i n a ting in com- position of Triplostegia was poorly resolved; pl e t e loss in Val e ri a n a c e a e . Fin a l l y,the Morin a c e a e - s u c c e s s ive - ap p rox i m ations weighting we a k ly Va l e ri a n a c e a e -Tri p l o s t egi a-Dipsacaceae cl a d e linked it with Valerianaceae. In our rbcL analy- is cha ra c t e ri z ed by distinctive che m i s t r y, in cl u d i n g sis it is we a k ly linked with Dipsacaceae the presence of alkaloids, ca thecolic tannins, instead.Taking morphological characters into and monoterpenoids. account (Backlund and Donog h u e, 1 9 9 6 ) , The link between the Linnaeeae and the Triplostegia is linked with both Valerianaceae M o ri n a c e a e - Va l e ri a n a c e a e -Tri p l o s t egi a- and Dipsacaceae on the basis of simple perfo- Dipsacaceae clade is also supported by several ration plates (or nearly so), further reduction of rather cl e a r-cut morp h o l ogical fe at u re s , a s the calyx lobes, several characters, and highlighted by Judd et al. (1994). num- the presence of ch l o ro p hyllous embryo s ber is reduced from five to four, which then (Yakovlev and Zhukova, 1980). Within this may be didynamous in arrangement. During clade, Triplostegia is united with Valerianaceae ovary development two of the three carpels on the basis of pollen morphological charac- abort, leaving a single-seeded carpel occupying t e rs , i n cluding ap e rt u res with a distinctive half of the ovary. Wilkinson (1949), in particu- “ h a l o ” ( B a cklund and Nilsson, 1 9 9 7 ) ; lar, stressed the great similarity of this condi- endosperm reduction in Triplostegia (Peng et tion in Linnaeeae and Valerianaceae, along al., 1995) or complete loss in Valerianaceae; with the similar median position and compound and chemical characters, especially the pres- vasculature of the ovule (also see Fukuoka, ence of va l ep o t ri ate iridoid compounds 1972). Similar carpel ab o rtion occurs in (Backlund and Moritz, 1996). Morinaceae and in Triplostegia. Under the view that this characterizes the entire clade, the loss Dipsacales monophyly. of a clear sign of ab o rted carpels in Several phylogenetic problems are not con- Dipsacaceae is interpreted as a further modifi- v i n c i n g ly re s o l ved by our analy s e s , m o s t cation. Finally, the ovary in all of these plants notably the question of the monophyly of tradi- matures into an . tional Dipsacales (Donoghue, 1983), including These results have especially import a n t just Adoxaceae and Caprifoliaceae sensu Judd implications for our understanding of ovary and et al. (1994). In most previous molecular stud- fruit evolution. Trees based on rbcL sequences ies, this clade has been recovered (Chase et al., alone indicate that Linnaeeae are not mono- 1993; Olmstead et al., 1992, 1993; Rice et al., phyletic and that Diervilleae were, in effect, 1 9 9 7 ) , though with only weak support . derived within Linnaeeae. Such trees imply that Donoghue et al. (1992) and Downie and Palmer the bi-carpellate, many-seeded, elongate cap- (1992) found alternative arrangements, some- sules ch a ra c t e ristic of the Diervilleae we re times including Araliaceae, but taxon sampling d e rived from the tri - c a rp e l l at e, o n e - s e e d e d, was limited in these studies. The main alterna- achene fruits seen in Linnaeeae, Morinaceae, tive was suggested by rbcL analyses conducted Tri p l o s t egi a,Va l e ri a n a c e a e, a n d, p ro b ably by Bremer et al. (1994), which implied that ancestrally, in Dipsacaceae. As we have noted, Desfontainia may be closely related to, or even this result is not upheld in combined analyses nested within, Dipsacales. Outgroups were too with morphological characters; Linnaeeae are poorly sampled by Judd et al. (1994) to provide instead linked with the Mori n a c e a e - a critical test using morp h o l ogical dat a . Valerianaceae-Triplostegia-Dipsacaceae clade. However, the Backlund and Donoghue (1996) Support for this result is not especially strong, morphological analysis, as well as their com- but it certa i n l y yields a simpler scenario for frui t bined analysis with rbcL, showed Columellia evolution. Specific a l l y, our combined trees imply and D e s fo n t a i n i a to be nested within that abortion of two carpels and achene fruits Dipsacales as the sister group(s) of the evol v ed once, at the base of the Linnaeeae-Mo r i- Caprifoliaceae clade. nac e a e - Va l e r ianaceae Trip l o s t e gia -D i p s a c a c e a e In our r b cL analysis the monophy ly of clade, and did not secondarily give rise to Dipsacales was supported, but only weakly bi-carpellate capsules. (bootstrap <50%; decay = 1); it requires just a 472 HARVARD PAPERS IN BOTANY Vol. 6, No. 2 single ex t ra step to move the C o l u m e l l i a- 1 0 4 , ch romosome size; 106, s t ru c t u ra l Desfontainia clade within the group. With the re a rra n gement of the ch l o roplast inve rt e d addition of the Backlund and Donoghue (1996) repeat; 108, size of the inverted repeat), and mo rp h o l o gical cha ra c t e rs , tr ees of both types are Columellia or Desfontainia lack the presumed equally parsimonious, that is, with Columellia derived state of 5 others (27, superficial phel- and De s fo n t a i n i a as the sister group of Dipsacales logen is not present in Desfontainia; 37, three and with these taxa nested within Dipsacales as vascular traces are not present in the calyx the sister group of Caprifoliaceae. lobes of Columellia; 46, corolla vasculature is The original Backlund and Donoghue (1996) not laterally connected in Columellia; 59, stig- analysis, which included a broader sample of mas are bilobed, not capitate, in Columellia, outgroups, appeared to show much better sup- and are polymorphic in Desfontainia; 69, the p o rt for the inclusion of C o l u m e l l i a a n d t apetum is glandular, not amoeb o i d, i n Desfontainia within Dipsacales. However, on Desfontainia). Of the remaining 5 characters, closer inspection, this is not the case. Both the vascular cylinder (21) was scored incorrectly clade containing the traditional Dipsacales plus for Viburnum and Sambucus (they do have Columellia and Desfontainia, and the branch cylinders); well-developed corolla tubes (42) linking C o l u m e l l i a and D e s fo n t a i n i a w i t h and stamens attached within (50) appear to be Caprifoliaceae, were very weakly supported directly correlated and are widespread among (bootstraps <50%; decay = 1). Furthermore, asterids; corolla zygomorphy (43) is polymor- reexamination of the morphological characters phic in most genera of Caprifoliaceae s.s.; and optimized as changing along these branches the production of cathecolic tannins (99) is (referred to below by character numbers used in poorly known and highly variable among pos- Backlund and Donoghue, 1996) shows that sible outgroups and must also be homoplastic most of these do not provide unequivocal sup- within Dipsacales. p o rt. The bra n ch including tra d i t i o n a l We conclude from this analysis that molecu- Dipsacales plus Columellia and Desfontainia is lar and morphological evidence is unconvinc- m a rked by 12 morp h o l ogical ch a n ges in ing that Columellia and Desfontainia are united Backlund and Donoghue (1996). Two of these with, or nested within, Dipsacales. These taxa are unknown in Columellia and Desfontainia— may be linked with Dipsacales on the basis of iridoids (character 87) and saponins (95)—and opposite leaves, fused , and tenuinucel- for 7 others the presumed derived state is not late ovules, and they may specifically be united p resent in C o l u m e l l i a and/or D e s fo n t a i n i a: with Caprifoliaceae on the basis of elongate l e aves are eve rgreen in C o l u m e l l i a a n d corolla tubes bearing the stamens. However, Desfontainia (4); leaf margins in Dipsacales these ch a ra c t e rs each show high levels of are very rarely spiny, as in Columellia and homoplasy among , and there are obvi- Desfontainia (11); leaf vernation is condupli- ous conflicting characters. Unlike traditional c ate in C o l u m e l l i a and D e s fo n t a i n i a ( 1 5 ) ; Dipsacales, Columellia and Desfontainia have Desfontainia has perforation plates with many spiny, evergreen leaves, bi-carpellate ovaries cross bars (24); rotate corollas are not found in that are half inferior in Columellia and fully C o l u m e l l i a and D e s fo n t a i n i a, or in superior in Desfontainia, solanad embryogeny, Caprifoliaceae (42); stamens are clearly fused and base chromosome numbers of X = 7. to the corolla in Columellia and Desfontainia, Placement within Dipsacales also seems odd as in Caprifoliaceae (50); chromosome num- from a biogeographic standpoint; traditional bers are X = 7 in both, not X = 8 as in Dipsacales are basically Northern Hemisphere Dipsacales (103). This leaves tenuinucellate in distribution,with greatest diversity in eastern ovules (65), opposite leaves (5), and petals Asia, whereas Columellia and Desfontainia are fused into a tube (40), all of which are wide- centered in South America. Finally, traditional spread among asterids, such that the location of Dipsacales do share a number of possible apo- character changes are heavily dependent on morphies, which may not have been correctly which outgroups are included in an analysis. represented in prior analyses, for example, the The bra n c h linking Co l u m e l l i a and De s fo n t a i n i a widespread occurrence of aborted ovules or with Caprifoliaceae is marked by changes in 14 vestigial archesporial tissue in the upper por- characters in Backlund and Donoghue (1996). tion of the ovary (see Erbar, 1994). Depending However, 4 of these characters are unknown in on details of broader relationships, cellular Columellia and Desfontainia (93, ellagic acid; endosperm development, asterad and related 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 473 forms of embryogeny, and 3- to 5-carpellate clade that includes A d oxaceae and gynoecia may also be synapomorphies of tradi- Caprifoliaceae. Although we consider it doubt- tional Dipsacales. ful on present evidence, we recognize the pos- sibility that the least inclusive clade including Phylogenetic Taxonomy A d oxaceae and Cap ri foliaceae might also It is possible to reflect our current under- include Columellia and Desfontainia. standing of Dipsacales phylogeny using tradi- With phyl o genetic nomenclat u r e, ne w names are tional Linnaean nomenclature; that is, using given only to clades that have not previ o u s l y been standard ranks such as family, tribe, and so on. na m e d . Within Ad ox a c e a e , we here propose the This ap p ro a ch has been taken by the name Ad o xina for the clade including Si n a d ox a , Angiosperm Phylogeny Group (APG, 1998), Tet ra d ox a , and Ad ox a . Within Capri f oliaceae we which recognized six families within an Order pr opose the name Val e r ina for the clade inclu d - Dipsacales that excludes Adoxaceae (which ing Morin a c e a e , Di p s a c a c e a e , Trip l o s t e gia , an d APG left unassigned within their “euasterid II” Val e ri a n a c e a e , and the name Linnina is app l i e d clade): (1) Caprifoliaceae (restricted to the for- to the clade including Val e r ina plus Linnaeeae. mer Caprifolieae), (2) Diervillaceae (for the For mal phyl o genetic definitions of these cla d e s former Diervilleae; Backlund and Pyck, 1998), ar e presented in Table 2. (3) Linnaeaceae (for the former Linnaeeae; Backlund and Pyck, 1998), (4) Morinaceae, (5) CONCLUSIONS Dipsacaceae, and (6) Valerianaceae (presum- P revious phy l ogenetic conclusions are ably including Tri p l o s t egi a; Backlund and l a rge ly confi rmed by our analy s e s . Bremer, 1998). Note that in this system old Caprifoliaceae in the traditional sense are not names are applied to clades that already have monophyletic, and there are two major lineages names (e.g., Caprifoliaceae for Caprifolieae; within Dipsacales: (1) Adoxaceae, including Dipsacales for Caprifoliaceae sensu Judd et al., Vibu r nu m, S a m bu c u s, S i n a d ox a, Te t ra d ox a, 1994), new names are introduced for clades and Adoxa; and (2) Caprifoliaceae, including that already have names that are in wide use C ap ri fo l i e a e, D i e rv i l l e a e, L i n n a e e a e, (Diervillaceae for Diervilleae, Linnaeaceae for Morinaceae,Valerianaceae, Triplostegia, and Linnaeeae), and, ironically, no new names are Dipsacaceae.Within Adoxaceae we conclude that Sinadoxa and Tetradoxa are linked with introduced to reflect widely accepted knowl- Adoxa, in Adoxina. Within Caprifoliaceae our edge of phylogenetic relationships among these combined analyses support Valerina, including groups (e.g., the link between Morinaceae, M o ri n a c e a e, D i p s a c a c e a e, Tri p l o s t egi a, a n d Valerianaceae, and Dipsacaceae, or between Valerianaceae, and Linnina, including Valerina this clade and Linnaeeae). plus Linnaeeae. Each of these clades is marked Phy l o genetic nomenclat u r e (se n s u de Queiroz by morphological synapomorphies. and Gauthier, 1992, 1994; see also Hibbett and Additional data are needed to test the mono- Donoghue, 1998), without ranks, deals with phyly of Linnaeeae, as well as to clarify the this problem more efficiently (Fig. 7). The re l ationships of Diervilleae (whether with names Cap ri fo l i e a e, D i e rv i l l e a e, L i n n a e e a e, C ap ri folieae or with Linnina). A l t h o u g h D i p s a c a c e a e, and Va l e rianaceae are simply H ep t a c o d i u m ap p e a rs to be re l ated to retained for their respective clades; renaming C ap ri folieae (rather than Linnaeeae), a n d these can only cause confusion. Following Judd Tri p l o s t egi a m ay be we a k ly united with et al. (1994), the name Cap ri foliaceae is Valerianaceae (rather than Dipsacaceae), we applied to the clade including all of these taxa. look forward to additional evidence on these Likewise, the name Adoxaceae is used for the issues. Relationships within Diervilleae have clade including Vi bu rnu m, S a m bu c u s, a n d recently been analyzed (Kim and Kim, 1999), Adoxa, and Adoxoideae is used for the clade but re l ationships within Cap ri folieae and including Sambucus and Adoxa (Donoghue, Linnaeeae remain poorly resolved. Finally, the 1983; Thorne, 1992; Eriksson and Donoghue question of the monophy ly of Dipsacales, 1997). On the basis of arguments presented whether including or excluding Columellia and above, the name Dipsacales is retained for the Desfontainia, needs further attention. 474 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

FIG U R E 7. Summary Dipsacales phylogeny showing the proposed application of names to clades. Phylogenetic definitions are provided in Table 2 for those clades marked with a dot. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 475

TAB L E 1. Vou c her inform a tion and GenBank accession num b e r s for species used in the rb c L and ITS analys e s .

SPECIES REFERENCE OR VOUCHER GENBANK NO. Abelia x grandiflora Rehder Cult. Univ. , Tucson, 1993; AJ420875 Donoghue, voucher lacking Adoxa moschatellina L. rbcL: Donoghue et al., 1992 L01884 ITS: Eriksson and Donoghue, 1997 U88194 Barnadesia caryophylla Olmstead et al., 1992 L01887 (Vell.) S. F. Blake Boopis anthemoides Juss. Olmstead et al., 1993 L13860 Brunia albiflora Phillips Backlund and Bremer, 1997 Y10674 Campanula ramulosa Wall. Olmstead et al., 1992 L13861 ruber (L.) DC. Cult. Univ. Arizona, Tucson, 1993; AJ420879 Donoghue, voucher lacking Columellia oblonga Ruiz & Pav. Backlund and Bremer, 1997 Y10675 Coriandrum sativum L. Olmstead et al., 1992 L11676 Cornus kousa Hance Olmstead et al., 1993 L14395 Desfontainia spinosa Ruiz & Pav. Bremer et al., 1994 Z29670 Buckley rbcL: Bremer et al., 1994 Z29672 ITS: Kim and Kim, 1999 AF078703 Dipelta floribunda Maxim. Cult. Arnold Arboretum, 14514-B; AJ420876 Kelly and Buckland 32 sativus (L.) Honck. Olmstead et al., 1992 L13864 Gentiana procera Holm Olmstead et al., 1993 L14398 Griselinia lucida G. Forst. Xiang et al., 1993 L11225 Hedera helix L. Olmstead et al., 1992 L01924 Heptacodium miconioides Rehder Cult. Arnold Arboretum 1549-80-D; AJ420873 Koller s.n., 12 Oct. 1984 (A) intermedia Pernh. & Wettst. Backlund and Bremer, 1997 Y10698 Kolkwitzia amabilis Graebn. Cult. Arnold Arboretum 18090; AJ420877 Elsik and Siegel 1558 (A) Wall. Cult. Kew Botanic Gardens, UK, 1990; AJ420872 Donoghue, voucher lacking L. Door County, WI, 1990; AJ420878 Donoghue, voucher lacking Lonicera orientalis Lam. Gustafsson et al., 1996 X87389 Lonicera prolifera (G.Kirchn.) Rehder Cult. Arnold Arboretum, 870-74-A; AJ420870 voucher lacking Menyanthes trifoliata L. Olmstead et al., 1993 L14006 Morina coulteriana Royle Backlund and Bremer, 1997 Y10706 jatamansii (D. Don) DC. Backlund and Bremer, 1997 Y10705 476 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

TABLE 1. (CONT.)

SPECIES REFERENCE OR VOUCHER GENBANK NO. Nicotiana tabacum L. Lin et al., 1986 Patrinia rupestris (Pall.) Dufr. Backlund and Bremer, 1997 Y10704 Phyllactis bracteata Wedd. Backlund and Bremer, 1997 Y10703 Pittosporum japonicum Morgan and Soltis, 1993 L11202 Hort. ex C. Presl. lasiospermus Backlund and Bremer, 1997 Y10702 Link ex Buch (Lindl.) Fritsch ITS: Eriksson and Donoghue, 1997 U41381 Cham. & Schltdl. ITS: Eriksson and Donoghue, 1997 U88196 Sambucus caerulea Rafinesque rbcL: vic.Tucson,Arizona, 1993; AJ420867 Donoghue, voucher lacking ITS: Eriksson and Donoghue, 1997 U88197 L. ITS: Eriksson and Donoghue, 1997 U88200 L. rbcL: Donoghue et al., 1992 L14066 ITS: Eriksson and Donoghue, 1997 U88207 Sinadoxa corydalifolia C. Y. Wu, rbcL: Boufford et al. 26555 (A) AJ420866 Z. L. Wu & R. F. Huang ITS: Boufford et al. 26555 (A) AJ419711 albus (L.) S. F. Blake Olmstead et al., 1992 L11682 Tetradoxa omeiensis rbcL: Donoghue et al. 4000 (A) AJ420865 (H. Hara) C. Y. Wu ITS: Donoghue et al. 4000 (A) AJ419710 L. vic. Madison, WI, 1990; AJ420870 Donoghue, voucher lacking Triplostegia glandulifera Wall. ex DC. Backlund and Bremer, 1997 Y10700 officinalis L. Olmstead et al., 1992 L13934 L. rbcL: Olmstead et al., 1992 L01959 ITS: Cult. Arnold Arboretum 1505-67; AJ420923 Elsik 2102 (A) AJ420924 L. ITS: Eriksson and Donoghue, 1997 U88552 U88553 L. ITS: Eriksson and Donoghue, 1997 U88554 U88555 Thunb. rbcL: Cult. Arnold Arboretum 1050-67-A; AJ420868 Dwyer et al., 4354 (A) ITS: Cult. Arnold Arboretum 1050-67-A; AJ420925 Dwyer et al., 4354 (A) AJ420926 Miq. rbcL: Cult. Arnold Arboretum 616-6-B; AJ420869 Elsik et al. 2640 (A) U88556 ITS: Eriksson and Donoghue, 1997 U88557 Weigela hortensis (Sieb. & Zuck.) rbcL: Cult. Arnold Arboretum 1897-77-A; AJ420874 C. A. Mey. Kelly and Buckland 28 (A) ITS: Kim and Kim, 1999 AF078713 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 477

TABLE 2. Phylogenetic definitions for clade names in Fig. 7.

CLADE DEFINITION1 NOTES Dipsacales L. Sensu Donoghue (1983) Linnaea borealis L. and Judd et al. (1994) L. Viburnum L. Adoxaceae L. Sensu Donoghue (1983) and Thorne L. (1983); defined phylogenetically by Adoxa moschatellina L. Judd et al. (1994) Adoxoideae Sambucus nigra L. Sensu Thorne (1983) Adoxa moschatellina L. Adoxina Adoxa moschatellina L. New clade name Tetradoxa omeiensis (H. Hara) C. Y. Wu Si n a d o xa coryd a l i fo l i a C. Y.Wu, Z. L. Wu & R. F. Huang Caprifoliaceae Lonicera caprifolium L Defined phylogenetically by Judd et al. Miller (1994); Dipsacales sensu APG (1998) Linnaea borealis L. Valeriana pyrenaica L. Dipsacus fullonum L. Caprifolieae Lonicera caprifolium L. Caprifoliaceae sensu Backlund and Pyck Symphoricarpos orbiculata Moench (1998) and APG (1998) Leycesteria formosa Wall. Diervilleae Diervilla lonicera Miller Diervilliaceae sensu Backlund and Pyck and Weigela japonica Thunb. (1998) and APG (1998) Linnina Abelia chinensis R. Brown New clade name Linnaea borealis L. Morina persica L. Valeriana pyrenaica L. Dipsacus fullonum L. Linnaeeae Abelia chinensis R. Brown Linnaeaceae sensu Backlund and Pyck Linnaea borealis L. (1998) and APG (1998) Kolkwitzia amabilis Graebner and Dipelta floribunda Maxim. Valerina Morina persica L. New clade name Valeriana pyrenaica L. Dipsacus fullonum L.

1 All of the definitions are "node-based" (de Queiroz and Gauthier, 1992, 1994; see the PhyloCode at http://www.ohiou.edu/phylocode/); each name is defined as the least-inclusive clade containing the taxa listed. 478 HARVARD PAPERS IN BOTANY Vol. 6, No. 2

LITERATURE CITED

ANGIOSPERM PHYLOGENY GROUP (APG). 1998. An DEVOS,F. 1951. The stem anatomy of some species ordinal classification for the families of flowering of the Caprifoliaceae with reference to phylogeny plants. Ann. Missouri Bot. Gard. 85: 531Ð553. and identification of the species. Ph.D. disserta- BACKLUND, A., AND B. BREMER. 1997. Phylogeny of tion, Cornell University, Ithaca, N.Y. the s. str. based on rbcL sequences, with DON O G H U E , M. J. 1983. The phyl o genetic rel at i o n - particular reference to the Dipsacales. Pl. Syst. ships of Vibu r num . Pages 143Ð166 in N. I. PLAT N I C K Evol. 207: 225Ð254. AN D V. A. FUN K , ED S ., Ad vances in Cladistics. Vol. 2. BACKLUND, A., AND K. B REMER. 1998. To be or not Columbia Univer sity Pres s , Ne w York . to be—principles of classification and monotypic DO N O G H U E, M. J. , A N D B. G. BA L DW I N. 1 9 9 3 . families. Taxon 47: 391Ð400. Phylogenetic analysis of Viburnum based on ribo- BACKLUND, A., AND M. J. DONOGHUE. 1996. Mor- somal DNA sequences from the internal tran- phology and phylogeny of the order Dipsacales. scribed spacer regions. Amer. Jour. Bot. 80: 146. Pages 1Ð55 in A. BACKLUND, ED., Phylogeny of the DONOGHUE, M. J.,R. H. REE, AND D. A. BAUM. 1998. Dipsacales. Part IV. Department of Systematic Phylogeny and the evolution of flower symmetry Botany, Uppsala University, Uppsala. in the Asteridae. Trends Plant Sci. 3: 311Ð317. BACKLUND, A., AND T. M ORITZ. 1996. Phylogenetic DONOGHUE, M. J., R. G. OLMSTEAD,J. F. SMITH, AND implications of an expanded valepotriate distribu- J. D. PALMER. 1992. Phylogenetic relationships of tion in the Va l e ri a n a c e a e. Pages 1Ð27 in A . Dipsacales based on rbcL sequences. Annals of BACKLUND, ED., Phylogeny of the Dipsacales. Part the Missouri Botanical Garden 79: 333Ð345. III. Department of Systematic Botany, Uppsala DOWNIE,S. R., AND J. D. PALMER. 1992. Restriction University, Uppsala, Sweden. site mapping of the chloroplast DNA inverted BACKLUND, A., AND S. NILSSON. 1997. Pollen mor- repeat: A molecular phylogeny of the Asteridae. p h o l ogy and the systematic position of Annals of the Missouri Botanical Garden 79: Triplostegia (Dipsacales). Taxon 46: 21Ð31. 266Ð283. ERBAR, C. 1994. Contributions to the affinities of BACKLUND, A., AND N. PYCK. 1998. Diervilliaceae and Linnaeaceae, two new families of caprifo- Adoxa from the viewpoint of floral development. lioids. Taxon 47: 657Ð661. Bot. Jahrb. Syst. 116: 259Ð282. ERIKSSON, T. 1999. AutoDecay. Version 4.0.2 (com- BALDWIN,B. G., M. J. SANDERSON,J. M. PORTER, M. puter program). Distributed by the author. Bergius F. WOJCIECHOWSKI, C. S. CAMPBELL, AND M. J. Foundation, Royal Swedish Academy of Sciences, DONOGHUE. 1995. The ITS region of nuclear ribo- Stockholm. somal DNA: A valuable source of evidence on ERIKSSON,T., AND M. J. DONOGHUE. 1997. Phyloge- angiosperm phylogeny. Annals of the Missouri netic re l ationships of S a m bu c u s and A d ox a Botanical Garden 82: 247Ð277. (Adoxoideae, Adoxaceae) based on nuclear ribo- BELL, C. D., E J. EDWARDS,S.-T. KIM, AND M. J. somal ITS sequences and preliminary morpholog- DONOGHUE. 2001. Dipsacales phylogeny based on ical data. Syst. Bot. 22: 555Ð573. chloroplast DNA sequences. Harvard Pap. Bot. 6: FELSENSTEIN,J. 1985. Confidence limits on phyloge- 481Ð499. nies: An approach using the bootstrap. Evolution BENKO-ISEPPON, A. M., AND W. MORAWETZ. 2000. 39: 783Ð791. Viburnales: Cytological features and a new cir- FUK U O K A , N. 1969. Inflo r escence of Linnaeae (Capr i- cumscription. Taxon 49: 5Ð16. foliaceae). Acta Phytotaxonomica Geobotanica BREMER,K. 1988. The limits of amino acid sequence 23: 153Ð162. data in angiosperm phylogenetic reconstruction. —— — . 1972. Taxonomic study of the Cap ri- Evolution 42: 795Ð803. foliaceae. Memoirs of the Faculty of Science, BREMER,B. R., G. OLMSTEAD, L. STRUWE, AND J. A. Kyoto University, Biology Series 6: 15Ð58. SWEERE. 1994. rbcL sequences support exclusion ———. 1974. Floral morp h o l ogy of A d ox a of R e t z i a, D e s fo n t a i n i a, and N i c o d e m i a mo s ch at e l l i n a . Acta Phytotaxonomica Geobotanica (B u d dlejaceae) from . Plant Systemati c s 26:65Ð76. and Evolution 190: 213Ð230. GUSTAFSON, M. G. H. , A. BACK L U N D , AN D B. BRE M E R . CAPUTO,G., AND S. COZZOLLINO. 1994. A cladistic 1996. Phylogeny of the Asterales sensu lato based a n a lysis of Dipsacaceae (Dipsacales). Plant on rbcL sequences with particular reference to Systematics and Evolution 189: 41Ð61. Goodeniaceae. Plant Syst. Evol. 199: 217Ð242. CHA S E , M. W., E TA L. (41 others). 1993. Phyl o gen e t i c s HARA, H. 1981. A new species of the Adoxa of plants: An analysis of nu cl e o t i d e from Mt. Omei of China. Journal of Japanese sequences from the plastid gene rbcL. Annals of Botany 56: 271Ð274. the Missouri Botanical Garden 80: 528Ð580. —— — . 1983. A Revision of the Caprifoliaceae of DE QUEIROZ, K., AND J. GAUTHIER. 1992. Phyloge- Japan with Reference to Allied Plants in Other netic taxonomy. Annual Review of Ecology and Districts and the Adoxaceae. Academia Scientific Systematics 23: 449Ð480. Books, Tokyo. —— — . 1994. Toward a phylogenetic system of bio- HI B B E T T, D. S. , A N D M. J. DO N O G H U E. 1 9 9 8 . l ogical nomencl at u re. Trends in Ecology and Integrating phylogenetic analysis and classifica- Evolution 9: 27Ð31. tion in fungi. Mycologia 90: 347Ð356. 2001 DONOGHUE ET AL., DIPSACALES PHYLOGENY 479

HOFMANN,U., AND J. G ÖTTMANN. 1990. Morina L. PYCK,N., P. ROELS, AND E. SMETS. 1999. Tribal rela- and Triplostegia Wall ex DC. im Vergleich mit tionships in Cap ri fo l i a c e a e : Evidence from a Va l e rianaceae und Dipsacaceae. Botanisch e cladistic analysis using ndhF sequences. Syst. Jahrbücher für Systematik Pflanzengeschichte und Geogr. Pl. 69: 145Ð159. Pflanzengeographie 111: 499Ð553. RA M BAU T, A . 1996. S e - A l : Sequence A l i g n m e n t JUDD,W. S., R. W. SANDERS, AND M. J. DONOGHUE. E d i t o r. Ve rsion 1.0a1 (computer progra m ) . 1994. Angiosperm family pairs: Preliminary phy- Distributed by the author. Department of Zoology, logenetic analyses. Harvard Pap. Bot. 5: 1Ð51. University of Oxford, Oxford, U.K. KIM,Y.-D., AND S.-H. KIM. 1999. Phylogeny of RICE, K. A., M. J. DONOGHUE, AND R. G. OLMSTEAD. Weigela and Diervilla (Caprifoliaceae) based on 1997. Analyzing large data sets: rbcL 500 revis- nuclear rDNA ITS sequences: Biogeographic and ited. Systematic Biology 46: 554Ð563. t a x o n o m i ci m p l i c at i o n s. J. Plant Res. 112: 33 1 Ð 3 4 1 . ROELS,P., AND E. SMETS. 1994. A comparative floral LAGERBERG, T. 1904. Organografiska studier öfver ontogenetical study between Adoxa moschatellina Adoxamoschatellina L. Arkiv for Botanik 3:1Ð28. and Sambucus ebulus. Belgian Journal of Botany LI, S. H., AN D Z. H. NIN G . 1987. Studies on the fam i l y 127: 157Ð170. Ad ox a c e a e .Bu l l e t i n of Bo t a n i c a l Re s e a r ch7:9 3 Ð 1 0 9. —— — . 1996. A floral ontogenetic study in the LI A N G, H . 1986. A n a lysis of the karyotype of Dipsacales. International Journal of Plant Sciences Tetradoxa omeiensis. Acta Botanica Yunnanica 8: 157: 203Ð218. 153Ð156. STADEN, R. 1996. The Staden sequence analysis —— — . 1997. A comparative study of floral vascu- package. Molecular Biotechnology. 5: 233Ð241. lature in Adoxaceae. Acta Botanica Yunnanica 19: SWO F F O R D, D. L. 2001. PAUP*. Phy l oge n e t i c 260Ð264. Analysis Using Parsimony (*and Other Methods). LIANG, H., AND X. ZHANG. 1986. Floral anatomy of Ve rsion 4 (computer program). Sinauer, Tetradoxa omeiensis. Acta Botanica Yunnanica 8: Sunderland, Mass. 436Ð440. THORNE, R. F. 1983. Proposed new realignments in LIN, C. M., Z.-Q. LIU, AND S. D. KING. 1986. the angiosperms. Nordic Jour. Bot. 3: 85Ð117. Nicotiana ch l o roplast ge n o m e : X. Corre l at i o n —— — . 1992. An updated classification of the flow- between the DNA sequences and the isoelectric ering plants. Aliso 13: 365Ð389. focusing pat t e rn of the LS of rubisco. Plant UR BAT S C H, L. E., B. G. BA L DW I N, A N D M. J. Molecular Biology 6: 81Ð87. DONOGHUE. 2000. Phylogeny of the coneflowers MADDISON,W. P., AND D. R. MADDISON. 1992. (Heliantheae:Asteraceae) based on nuclear rDNA MacClade, Analysis of Phylogeny and Character internal transcribed spacer sequences. Syst. Bot. Evolution. Version 3 (computer program). Sinauer, 25: 539Ð565. Sunderland, Mass. VE R L AQU E, R . 1977. Rap p o rts entre les MANCHESTER,S. R., AND M. J. DONOGHUE. 1995. Valerianaceae, les Morinaceae et les Dipsacaceae. Winged fruits of Linnaeeae (Caprifoliaceae) in the Bull. Soc. Bot. France 124: 475Ð482. Tertiary of Western : Diplodipelta WAGENITZ,G., AND B. LAING. 1984. Die Nektarien gen. nov. International Journal of Plant Science der Dipsacales und ihre systematische Bedeutung. 156: 709Ð722. Bot. Jahrb. Syst. 104: 91Ð113. NEPOMNYASHCHAYA, O. A. 1984. On a new species of WEBERLING, F. 1989. Morphology of flowers and the genus Adoxa (Adoxaceae) from the far east. i n fl o rescences. Cambri d ge Unive rsity Pre s s , Botanicheskii Zhurnal 69: 259Ð262. Cambridge, U.K. OLM S T E A D , R. G., B. BRE M E R , K. M. SCOT T , AN D J. D. WILKINSON, A. M. 1948. Floral anatomy and mor- PAL M E R . 19 9 3. A parsi m o n y analysis of the As t e ri d a e phology of the genus Viburnum. American Journal sensu lato based on rbcL sequences. Annals of the of Botany 35: 455Ð465. Missouri Botanical Garden 80: 700Ð722. —— — . 1949. Floral anatomy and morphology of OLMSTEAD, R. G.,H. J. MICHAELS, K. M. SCOTT, AND Triosteum and of the Caprifoliaceae in general. J. D. PALMER. 1992. Monophyly of the Asteridae American Journal of Botany 36: 481Ð489. and identification of their major lineages inferred WU, C. Y. 1981. Another new genus of Adoxaceae, from DNA sequences of rbcL. Annals of the with special references on the infrafamiliar evolu- Missouri Botanical Garden 79: 249Ð265. tion and the systematic position of the family. Acta OLMSTEAD, R. G., K.-J. KIM, R. K. JANSEN, AND S. J. Botanica Yunnanica 3: 383Ð388. WAGSTAFF. 2000. The phylogeny of the Asteridae WU, C. Y., Z. L. WU, AND R. F. HUANG. 1981. s e n s u l at o based on ch l o roplast n d hF ge n e Sinadoxa C. Y.Wu,Z. L. Wu et R. F. Huang, genus sequences. Mol. Phylo. Evol. 16: 96Ð112. novum familiae Adoxacearum. Acta Phytotax. Sin. PENG, C.-I., H. TOBE, AND M. TAKAHASHI. 1995. 19: 203Ð210. Reproductive morphology and relationships of XIANG,Q.-Y., D. E. SOLTIS,P. S. SOLTIS, AND D. R. Triplosetgia (Dipsacales). Bot. Jahrb. Syst. 116: MO R G A N. 1993. Phy l ogenetic re l ationships of 505Ð516. C o rnu s L. sensu lato and putat ive re l at ive s PYCK,N., AND E. SMETS. 2000. A search for the phy- inferred from rbcL sequence data. Annals of the l ogenetic position of the seven-son fl owe r Missouri Botanical Garden 80: 723Ð734. (Heptacodium, Dipsacales): Combining molecular YA KOV L E V, M. S. , A N D G. Y. ZH U KOVA. 1 9 8 0 . and morphological evidence. Plant. Syst. Evol. Chlorophyll in embryos of angiosperm , a 225: 185Ð199. review. Botaniska Notiser 133: 323Ð336.