DIPSACALES PHYLOGENY BASED ON CHLOROPLAST DNA SEQUENCES CHARLES D. BELL,1, 2 ERIKA J. EDWARDS,1 SANG-TAE KIM,1 AND MICHAEL J. DONOGHUE 1 Abstract. Eight new rbcL DNA sequences and 15 new sequences from the 5' end of the chloroplast ndhF gene were obtained from representative Dipsacales and outgroup taxa. These were analyzed in combination with pre- viously published sequences for both regions. In addition, sequence data from the entire ndhF gene, the trnL-F intergenic spacer region,the trnL intron,the matK region, and the rbcL-atpB intergenic spacer region were col- lected for 30 taxa within Dipsacales. Phylogenetic relationships were inferred using maximum parsimony and maximum likelihood methods. Inferred tree topologies are in strong agreement with previous results from sep- arate and combined analyses of rbcL and morpholo gy, and confidence in most major clades is now very high. Concerning controversial issues, we conclude that Dipsacales in the traditional sense is a monophyletic group and that Triplostegia is more closely related to Dipsacaceae than it is to Valerianaceae. Heptacodium is only weakly supported as the sister group of the Caprifolieae (within which relationships remain largely unresolved), and the exact position of Diervilleae is uncertain. Within Morinaceae, Acanthocalyx is the sister group of Morina plus Cryptothladia. Dipsacales now provides excellent opportunities for comparative studies, but it will be important to check the congruence of chloroplast results with those based on data from other genomes. Keywords: Dipsacales, Adoxaceae, Caprifoliaceae, Morinaceae, Dipsacaceae, Valerianaceae, phylogeny, chloroplast DNA. The Dipsacales has traditionally included the Linnaea). It excludes Adoxa and its relatives, as C ap ri foliaceae (s e n s u l at o, i . e. , i n cl u d i n g well as Dipsacaceae, M o ri n a c e a e, a n d Viburnum and Sambucus), Adoxaceae, Dip- Valerianaceae. We reject this traditional cir- sacaceae, and Valerianaceae (e.g., Cronquist, cumscription of Caprifoliaceae on the grounds 1 9 8 8 ) , and sometimes segregates such as of non-monophyly. According to all recent phy- Morinaceae and Triplostegiaceae. In contrast, logenetic studies (e.g., Donoghue, 1983, 1985; the classification of the Angiosperm Phylogeny D o n oghue et al., 1992; Ju dd et al., 1 9 9 4 ; Group (APG, 1998) included the bulk of the Backlund and Donoghue, 1996; Backlund and C ap ri foliaceae in Dipsacales, along with Bremer, 1997; Kim et al., 1999; Olmstead et Dipsacaceae, Valerianaceae, and Morinaceae, al., 2000; Donoghue et al., 2001), Sambucus but exc luded an expanded Ad o xaceae (inclu d i n g and Vibu r nu m a re more cl o s e ly re l ated to Viburnum, Sambucus, and Adoxa), which they A d ox a and its re l at ives than they are to left unassigned to any order within Asteridae. C ap ri fo l i e a e, D i e rv i l l e a e, and Linnaeeae, The molecular phylogenetic studies reported wh i ch are instead more cl o s e ly re l ated to he r e concern Dipsacales in the traditional sense. Morinaceae, Dipsacaceae, and Valerianaceae. The circu m s c r iption of the Capri f oliaceae has A more restricted usage of Caprifoliaceae also become confusing, and to avoid misunder- ex cludes S a m bu c u s and Vi bu rnu m, on the standings we need to clarify our usage of the grounds that these are probably more closely name. Caprifoliaceae in the traditional sense related to Adoxa and its relatives, but retains i n cludes S a m bu c u s, Vibu r nu m, C ap ri fo l i e a e C ap ri fo l i e a e, D i e rv i l l e a e, and Linnaeeae. (Leycesteria, Lonicera, Symphoricarpos, and Again, we reject the use of Caprifoliaceae in Tri o s t e u m, and possibly H ep t a c o d i u m) , this restricted sense because it is not mono- D i e rvilleae (D i e rv i l l a and We i ge l a) , a n d phyletic. Specifically, all recent analyses (refer- Linnaeeae (Abelia, Dipelta, Kolkwitzia, and ences above) indicate that the Linnaeeae is We are grateful to Torsten Eriksson, Pat Reeves, and Dick Olmstead for generating a number of the sequences used in our analyses and for allowing us to refer to their manuscript with MJD. We also acknowledge Kris Peterson for her help with sequencing. This study has most recently been supported by an NSF Biotic Surveys and Inventories grant to collect plants in the Hengduan Mountain region of China (DEB-9705795). 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut,06511-8106, U.S.A. 2 Author for correspondence. E-mail: [email protected]. Harvard Papers in Botany, Vol. 6,No. 2, 2001, pp. 481–499. © President and Fellows of Harvard College, 2001. 482 HARVARD PAPERS IN BOTANY Vol. 6, No. 2 m o re cl o s l ey re l ated to Mori n a c e a e, D i p- Adoxa clade, and the remainder of the tradi- s a c a c e a e, and Va l e ri a n a c e a e, than to the tional Cap ri foliaceae are parap hy l e t i c. Caprifolieae or Diervilleae. Specifically, the Linnaeeae appears to be more In view of the phylogenetic results, and wish- closely related to Morinaceae, Valerianaceae, ing to retain monophyly, Backlund and Bremer and Dipsacaceae than it is to either Caprifolieae (1998) suggested another strat egy, wh i ch or Dierv i l l e a e. Howeve r, s u p p o rt for these Backlund and Pyck (1998) followed with the major clades varies considerably among analy- naming of two new families. They restricted the ses, and the exact placement of several key taxa name Cap ri foliaceae to the fo rmer Cap ri- ( e. g. , H ep t a c o d i u m, Tri p l o s t egi a) re m a i n s fo l i e a e, and then elevated Diervilleae to unresolved. Moreover, there is disagreement Diervillaceae and Linnaeeae to Linnaeaceae. concerning the placement of several other taxa, This usage was adopted in the Angiosperm es p e c i a l l y Li n n a e a and the Diervi l l e a e , as well as Phylogeny Group ordinal classification (APG, relationships within the Caprifolieae. Finally, 1998). We reject this strategy on the grounds s eve ral lineages have re c e ived insuffi c i e n t that it causes instability and confusion to sub- attention. For instance, we still know little stitute the names Caprifoliaceae, Diervillaceae, about relationships among Morina, Acantho - and Linnaeaceae for the already well estab- calyx, and Cryptothladia of the Morinaceae. lished names Cap ri fo l i e a e, D i e rv i l l e a e, a n d Here we present broad phylogenetic analyses Linnaeeae solely for the sake of adjusting taxo- including 8 new rbcL sequences and 15 new nomic ranks. These names refer to the same DNA sequences from the 5' end of the ndhF entities, and rank assignments are arbitrary gene. For more detailed studies within Dip- (APG, 1998). sacales we have added sequences of several Our usage of both the names Adoxaceae and other ch l o roplast coding and non-coding Caprifoliaceae follows the initial suggestion of regions: matK, the trnL intron, and the inter- Judd et al. (1994) and the node-based phyloge- genic spacer (IGS) regions of trnL-F and rbcL- netic definitions of Donoghue et al. (2001). The atpB. Our hope was that separate and combined mapping of these names onto a phylogenetic analyses of this much-expanded chloroplast hypothesis is shown in Fig. 1, adapted from d ataset would further cl a rify Dipsacales Donoghue et al. (2001). Adoxaceae here refers phylogeny. to the clade that includes Viburnum, Sambucus, and Adoxa and its relatives. Caprifoliaceae MATERIALS AND METHODS includes Caprifolieae, Diervilleae, Linnaeeae, Samples Morinaceae, Dipsacaceae, and Valerianaceae. We carried out analyses on two different The unusual and most obviously beneficial fea- datasets. Our 46-taxon dataset included rbcL ture of this nomenclature is that all of the tradi- and ndhF sequences from 32 representatives of tional names are retained (so long as these refer major lineages within Dipsacales and 14 asterid to clades). The potentially confusing aspect of outgroup taxa. Our 30-taxon dataset consisted this treatment is that, in addition to including of Dipsacales taxa alone scored for all six Caprifolieae, Diervilleae, and Linnaeeae, the chloroplast gene regions. Information on the C ap ri foliaceae also includes Mori n a c e a e, source of plant materials and on GenBank Dipsacaceae, and Valerianaceae. We believe accession num b e r s is provided in Tables 1 and 2. that the benefits of this solution far exceed any potential confusion. Donoghue et al. (2001) DNA Extraction and PCR Protocols provide additional discussion of this approach. Total DNAs were extracted using the CTAB Previous analyses of Dipsacales phylogeny method of Doyle and Doyle (1987). The DNA have been based on morphological characters, extracts were further purified with the Prep-A- molecular data, and a combination of the two Gene DNA Purification Kit (Bio-Rad). Double- (e.g., Donoghue, 1983; Donoghue et al., 1992; stranded copies of all regions were amplified Judd et al., 1994; Backlund and Donoghue, using standard Po ly m e rase Chain Reaction 1996; Backlund and Bremer, 1997; Pyck et al., (PCR) in 25- to 50-µL reactions.