the phylogeny of the asteridae sensu late based on chloroplast ndhf gene sequences

The Phylogeny of the Asteridae sensu lato Based on

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Molecular Phylogenetics and EvolutionVol. 16, No. 1, July, pp. 96–112, 2000doi:10.1006/mpev.1999.0769, available online at http://www.idealibrary.com on

Chloroplast ndhF Gene SequencesRichard G. Olmstead,* Ki-Joong Kim,†,1 Robert K. Jansen,† and Steven J. Wagstaff‡

*Department of Botany, University of Washington, Seattle, Washington 98195; †Section of Integrative Biology andInstitute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712; and ‡Manaaki Whenua,

Landcare Research, P.O. Box 69, Lincoln 8152, New Zealand

Received July 23, 1999; revised December 21, 1999

likely to represent synapomorphies for the group (Olm-s

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A phylogenetic study of Asteridae sensu lato wasconducted based on chloroplast ndhF gene sequencesfor 116 ingroup and 13 outgroup species. Prior molec-ular studies based on rbcL sequences identified termi-nal groups corresponding to families, but were unableto resolve relationships among them. These results arelargely consistent with earlier rbcL studies, but pro-vide much greater resolution and stronger bootstrapsupport throughout the tree. The parsimony analysisfound eight equally parsimonious trees, all of whichrecognize four major clades with the following rela-tionship: (Cornales (Ericales (Euasterids I, EuasteridsII))). Euasterids I includes (Garryales ((Solanales, Bor-aginaceae) (Gentianales, Lamiales))), although withweak support for relationships among the namedclades. Euasterids II includes (Aquifoliales (Asterales(Apiales, Dipsacales))) with strong support for theserelationships. Relationships within Ericales areweakly supported and merit further attention. © 2000

Academic Press

Key Words: Asteridae; phylogeny; ndhF; angiospermclassification.

INTRODUCTION

A growing body of molecular evidence has accrued tosupport the recognition of a major lineage of eudicotangiosperms called the Asteridae sensu lato (Olmsteadet al., 1992, 1993; Chase et al., 1993; Soltis et al., 1997).

he Asteridae sensu lato (henceforth simply called As-eridae) includes the families placed in the subclass ofhe same name by Cronquist (1981), plus many otheramilies in his system assigned to subclasses Dilleni-dae, Hamamelideae, and Rosideae, and comprises ap-roximately one-third of all angiosperm species. Thisroup is characterized predominantly by the presencef unitegmic and tenuinucellar ovules and the wide-pread occurrence of iridoid compounds, which seem

1 Present address: Department of Biology, Young-Nam University,Kyung San City, Kyung Buk, South Korea.

1055-7903/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

96

tead et al., 1993). Another common and widespreadcharacteristic shared by members of this group is theoccurrence of fused corollas. Ontogenetic studies haveshown that there are two developmental paths to floralfusion, “early sympetaly” and “late sympetaly” (Erbarand Liens, 1996). Polypetalous representatives exam-ined exhibit early sympetaly, suggesting that corollafusion may be another synapomorphy of the group(Erbar and Liens, 1996).

Initial molecular systematic studies of the Asteridaebased on rbcL sequences succeeded in circumscribing thegroup and identifying its major lineages (Olmstead et al.,1992, 1993; Chase et al., 1993). However, the degree ofresolution among those lineages was limited and thestrength of support for some of the identified groups wasweak. It was clear that additional data would be neededto confirm the monophyly of the lineages identified byrbcL studies, to determine the relationships among thoselineages, and to make recommendations for changes inclassification that reflect our current understanding ofthe phylogeny of this group.

This study of Asteridae phylogeny based on sequencesof the chloroplast gene ndhF is parallel to those based onrbcL. Initial applications of ndhF sequences to systematictudies centered on relationships within families, makingse of its greater length and higher average substitutionate to generate ca. three times as many parsimony-nformative characters (Olmstead and Sweere, 1994;cotland et al., 1995; Clark et al., 1995; Kim and Jansen,995; Olmstead and Reeves, 1995; Scotland et al., 1995;eyland and Urbatsch, 1996; Catalan et al., 1997; Terry

t al., 1997a,b). As more sequences from different familiesecame available, it became apparent that ndhF se-uences would have application to phylogenetic ques-ions at much greater phylogenetic depth than justithin families (Olmstead et al., 1998). The sampling for

his study is similar to that of previous rbcL studies withrespect to family diversity, but is not universally parallelat the generic level; therefore, combining these data with

data from previous studies is inadvisable until consider-

qsprll

s1a

Jansen lab (and for some sequences in the Olmstead lab),

97CHLOROPLAST PHYLOGENY OF THE ASTERIDAE

ably more work is done to fill in gaps in both data sets.The goals of this study were to corroborate the re-

sults of prior molecular studies based on rbcL se-uences and to achieve greater resolution of relation-hips among groups within Asteridae. The resultsresented here (along with many other studies) al-eady have contributed to a newly proposed, ordinal-evel classification of angiosperms (Angiosperm Phy-ogeny Group, 1998).

MATERIALS AND METHODS

This study includes ndhF sequences for 129 angio-perm species, including 116 species of Asteridae s.l. and3 outgroup species (Appendix 1). Many of the plantccessions and 35 of the ndhF sequences have been used

in previously published molecular systematic studies,however, 94 sequences are first reported in this study(see Appendix 1 for GenBank Accession Nos.).

PCR and DNA sequencing methods were as describedin Olmstead and Sweere (1994), Olmstead and Reeves(1995), and Kim and Jansen (1995). Initial sequencealignments were constructed with the assistance of thecomputer program CLUSTAL W (Thompson et al., 1994)and adjusted by eye. Gaps shared by two or more taxawere scored as additional binary characters. In cases inwhich gaps of different length shared a start or end pointor in cases in which one gap entirely overlapped the otherin different taxa, the character defined by the shorter gapwas scored as unknown for the taxon with the longer gap(see Table 1 for further information about individual gapcharacters).

Parsimony analyses were conducted with all nucleo-tide and gap changes weighted equally using the com-puter program PAUP* 4.0 (vers. b2, Swofford, 1999). Atotal of 100 replicate tree searches was undertaken, eachwith a random taxon addition starting tree with TBRbranch swapping and MULTREES on. Subsequent tothese searches, 500 replicate heuristic searches were un-dertaken using the strict consensus tree of the most-parsimonious trees as a constraint, so that only thosetrees that did not match the constraint were retained inan effort to find shorter or other equal-length trees (Cata-lan et al., 1997; Rice et al., 1997). Bootstrap analysis with500 replicates was carried out using a heuristic searchwith TBR branch swapping and MULTREES off (DeBryand Olmstead, 2000). Also, a bootstrap analysis of therbcL data set of Olmstead et al. (1993) was conductedusing the same parameters as for the ndhF analysis toprovide bootstrap support values for comparison betweenstudies for clades found by both.

RESULTS

The PCR strategies used by the two collaborating labsdiffered in the proportion of the ndhF gene sequenceobtained. Primers external to the gene were used in the

so that the entire sequence of the gene was obtained (Kimand Jansen, 1995), whereas internal primers locatednear the ends of the gene were used for most taxa in theOlmstead lab, so that 23 nucleotides (nt) at the 59 end ofthe gene and 108 nt at the 39 end of the gene were notobtained for many sequences (Olmstead and Sweere,1994; Olmstead and Reeves, 1995). A 152-nucleotide re-gion in the middle of the Hydrostachys sequence couldnot be aligned readily with the other sequences, so wasexcluded from analysis. These regions and occasionalmissing data points in the middle of sequences accountfor approximately 2% missing data. The aligned se-quences begin at the start codon and end at the locationof the stop codon in Asteraceae (6 nt before the stop codonin Nicotiana and many other taxa). The total alignedsequence length, including gaps, was 2454 nucleotides.Of these, 785 were invariant among the sampled taxa,319 were variable but uninformative for parsimony anal-ysis, and 1350 were parsimony informative. Of the infor-mative positions, 417 occupied first codon positions, 306were second codon positions, and 627 were third codonpositions, A total of 82 gaps were inserted to align thesequences (Table 1), including 24 shared by two or moretaxa, which were included in the data set as binary char-acters, making a total of 1374 cladistic characters.

The strategy for parsimony analysis enabled the dis-covery of multiple islands. A total of eight trees repre-senting two islands of four trees each was recovered withlengths of 13,379 steps (CI 5 0.251; RI 5 0.534). Noshorter trees or additional trees at the same length werefound using the inverse constraint procedure, lendingfurther support to the inference that the strict consensustree of the eight trees (Fig. 1) encompasses the range ofpossible most-parsimonious tree topologies, given thesedata. One of the most-parsimonious trees is depicted inFig. 2 to illustrate representative branch lengths (usingaccelerated transformation optimization, ACCTRANS, inPAUP*). The gap characters, though few in number,were found to be more highly consistent with the shortesttree topology than were the nucleotide characters (CI 50.600 vs 0.250; RI 5 0.882 vs 0.532). Gaps provide partialsupport for 19 clades, 16 of which have bootstrap valuesof 97% or greater. Among the nucleotide characters, con-sistency with a representative shortest tree depicted inFig. 2 was greater on average among second codon posi-tions (CI 5 0.303; RI 5 0.551) than among first (CI 50.286; RI 5 0.521) or third (CI 5 0.213; RI 5 531) codonpositions.

For comparison with rbcL, in the study of Olmsteadet al. (1993), in which 105 rbcL sequences were ana-lyzed, representing a phylogenetic distribution verysimilar to that represented here, there were only 495parsimony-informative nt characters and no gap char-acters, or only 36% as many characters as ndhF pro-vided for this study.

TABLE 1

G

98 OLMSTEAD ET AL.

Asteridae ndhF Sequence Alignment Gaps

apaCharacter

No.b Location Sizec Polarity Taxa

1 657/658 6 Insertion Codonanthe2A 1371–1394 24 Deletion Hydrolea3A 1393–1398 6 Deletion Dicentra4A 1 1396–1404 9 Deletion Acanthopanax, Hedera, Panax5A 2 1401/1402 6 Insertion Cephalanthus, Guettarda, Rogiera, Erithalis6A 1401–1403 3 Deletion Pentaphragma7A 1415–1420 6 Deletion Anagallis8A 1426/1427 6 Insertion Selago9A 1439/1440 3 Insertion Gelsemium

10A 1440/1441 6 Insertion Montinia11A 3 1448/1449 9 Deletion Aquilegia, Nicotiana, Lycopersicon, Nolana,

Petunia, Schizanthus12 1449–1452 12c Deletion Impatiens13 1449–1488 42c Deletion Callitriche14 1460/1461 3 Insertion Sambucus15 1462/1463 3 Insertion Pentas16 1467–1475 15 Deletion Anagallis17 41 1467/1468 9 Insertion Garrya, Aster, Helianthus, Lactuca, Gerbera,

Dasyphyllum, Barnadesia, Boopis, Dampiera,Scaevola, Villarsia, Nymphoides, Menyanthes,Corokia, Pentaphragma, Trachelium,Campanula, Codonopsis, Cyananthus,Lobelia, Stylidium, Scabiosa, Dipsacus,Succisa, Valeriana, Abelia, Lonicera,Symphoricarpus, Sambucus, Viburnum,Angelica, Coriandrum, Pittosporum,Acanthopanax, Hedera, Panax, Griselinia,Ilex, Helwingia, Phyllonoma, Rhododendron,Impatiens, Diospyros, Phlox, Anagallis,Halesia, Styrax, Camellia, Hydrostachys,Carpenteria, Hydrangea, Eucnide, Cornus,Alangium, Heuchera, Cercidiphyllum,Rhamnus, Quercus, Acer, Paeonia

18 1467/1468 3 Insertion Aquilegia19B 51 1468–1475 9 Deletion Hellwingia, Phyllonoma, Griselinia, Osmorhiza20B 1470–1478 9 Deletion Borago21B 1470/1471 6 Insertion Selago22B 1475/1476 15 Insertion Phytolacca23B 61 1479/1480 6 Insertion Ilex, Eucnide24B 71 1476–1484 9 Deletion Gentiana, Exacum, Pentas25B 1482–1484 3 Deletion Gelsemium26B 1485/1486 6 Insertion Phlox27B 82 1486–1488 3 Deletion Veronica, Plantago, Digitalis, Carpenteria,

Eucnide28B 1486–1491 6 Deletion Lobelia29B 9 1488–1490 3 Deletion Ilex, Helwingia, Phyllonoma30B 1489–1494 6 Deletion Veronica31B 1494/1495 6 Insertion Acer32B 1495–1500 6 Deletion Justicia33B 10 1498–1506 9 Deletion Gentiana, Exacum34B 1498–1509 12 Deletion Ipomoea35B 11 1504–1506 3 Deletion Clerodendrum, Tetraclea, Ajuga36B 1504–1509 6 Deletion Impatiens37B 12 1506–1514 9 Deletion Sambucus, Viburnum38B 1513–1521 9 Deletion Boopis39B 1515/1516 21 Insertion Schizanthus40B 1518–1526 9 Deletion Paeonia41B 133 1519/1520 6 Insertion Phlox, Phytolacca42B 1525–1590 66 Deletion Ipomoea43B 1527/1528 3 Insertion Rhododendron44B 1533/1534 3 Insertion Quercus45B 1534–1536 3 Deletion Spigelia

TABLE 1—Continued

G

5

5

6

77

1

dc


apaCharacter

No.b Location Sizec Polarity Taxa

46B 1535/1536 6 Insertion Sambucus47B 144 1544/1545 6 Insertion Clerodendrum, Tetraclea, Ajuga, Vitex, Mentha,

Veronica, Digitalis, Callitriche, Justicia,Thunbergia, Phyla, Lantana, Aloysia,Sesamum, Buddleja, Paulownia,Scrophularia, Verbascum, Selago, Myoporum,Campsis, Tecoma, Catalpa, Retzia,Euthystachys, Codonanthe, Nematanthus,Drymonia, Cyrtandra

48B 1544/1545 18 Insertion Cephalanthus49B 1546–1551 6 Deletion Cyananthus50B 1549–1554 6 Deletion Acer51B 1551/1552 9 Insertion Rhamnus52B 1557/1558 6 Insertion Vitex53B 1558–1560 3 Deletion Cyrtandra54B 1560/1561 6 Insertion Succisa55B 1561–1566 6 Deletion Cercidiphyllum56B 155 1567–1569 6c Deletion Liriodendron, Magnolia7B 164,5 1569/1570 6 Insertion Gentiana, Exacum

58B 6 1569/1570 3 Insertion Hydrostachys (in hypervariable region notincluded in analysis)

9B 1581/1582 6 Insertion Dicentra60 1585–1587 3 Deletion Staphylea61C,D 1603–1608 6 Deletion Hydrolea62E 1686–1688 3 Deletion Ipomoea63F 177 1687/1688 9 Insertion Plantago, Codonanthe, Nematanthus, Drymonia64E 18 1690–1698 9 Deletion Montinia, Hydrolea5E 1691–1699 9 Deletion Mentha

66E 198 1691/1692 6 Insertion Dipsacus, Scabiosa, Succisa67E 208 1693–1698 6 Deletion Trachelium, Campanula, Codonopsis,

Cyananthus, Lobelia68E,F 1695/1696 6 Insertion Gentiana69E,F 21 1699–1707 9 Deletion Nyctanthes, Jasminum, Olea, Ligustrum0E,F 22 1700–1708 9 Deletion Aster, Helianthus, Lactuca1E,F 1701/1702 6 Insertion Phyllonoma

72E,F 1703/1704 6 Insertion Plantago73E,F 239 1703–1708 6 Deletion Hydrophyllum, Phacelia, Borago74E,F 1705–1710 6 Deletion Justicia75G 1743–1766 24 Deletion Pentaphragma76G 1759–1764 6 Deletion Ipomoea77 1878–1886 9 Deletion Lactuca78 1904/1905 6 Insertion Aquilegia79 1906/1907 6 Insertion Pentas80 1911/1912 6 Insertion Camellia81 1912/1913 6 Insertion Justicia82 24 1926/1927 6 Insertion Rhamnus, Quercus, Acer

Note. Location of gaps is in reference to the ndhF sequence of Nicotiana tabacum (Shinozaki et al., 1986; as corrected by Olmstead et al.,993). A “–” indicates the range of nucleotides deleted and a “/” indicates the location of an insertion relative to the sequence of Nicotiana.

Character number refers to the phylogenetic analysis, wherein gaps are coded 0/1 for absence and presence of sequence, respectively. Sizesof deletions that overlap insertions in the alignment for other taxa are exclusive of the insertion length, to indicate the actual size of thedeletion.

a Missing sequence in the region of these gaps as follows: A, Stylidium; B, Hydrostachys (the complete sequence for Hydrostachys has beenetermined through this region, positions 1468–1582 in Nicotiana, but alignment difficulties make coding this region as missing aonservative choice); C, Callitriche; D, Aquilegia; E, Exacum; F, Rhododendron; G, Dicentra.

b Character scoring for taxa with completely overlapping gaps is as follows: 1, missing for Callitriche due to overlap with deletion 13; 2,missing for Callitriche and Lobelia due to overlap with deletions 13 and 29, respectively; missing for Hydrostachys, due to unalignable regionin Hydrostachys; 3, missing for Boopis and Paeonia due to overlap with deletions 38 and 40, respectively; 4, missing for Ipomoea due tooverlap with deletion 42; 5, missing for Liriodendron and Magnolia, due to overlap with 56; 6, for the complete sequence of Hydrostachys,the best fit alignment (not included in the PAUP data set) suggests a 3-nt insertion somewhere in the 6-nt gap in 37; 7, missing for Ipomoea,due to overlap with deletion 62; 8, missing for Montinia and Hydrolea, due to overlap with deletion 64; 9, missing for Aster, Helianthus, andLactuca, due to overlap with 70.

c Includes a portion of a gap in Nicotiana.

100 OLMSTEAD ET AL.

DISCUSSIONThese results agree with those of previous cpDNA

studies in finding a monophyletic Asteridae (Olmstead

FIG. 1. Strict consensus tree of eight most-parsimonious trees bsupport values (numbers in parentheses in (a) represent values whePhylogeny Group (1998) ordinal classification. Shaded boxes encloEricales, and Euasterids II. (b) Euasterids I.

et al., 1992, 1993; Chase et al., 1993), although theoutgroup sampling was not designed for a rigorous testof this hypothesis. Alignment of the most remote out-

d on ndhF sequences. Numbers above branches indicate bootstrapydrostachys is not included). Classification follows the Angiosperm

the Euasterid I and Euasterid II clades. (a) Outgroups, Cornales,

asen Hse

t(goCdC


group sequences with the rest of the sequences wasproblematic in places, but alignment of outgroup se-quences closest to Asteridae was straightforward. Forthis reason, some of the outgroup relationships may bequestionable (although relationships among outgroupsare consistent with rbcL results wherever bootstrapvalues are .50%), but they should be sufficient to root

FIG. 1—

he asterid portion of the tree. Sequences of two taxaIpomoea and Hydrostachys) were particularly diver-ent. Ipomoea had four unique deletions, including onef 66 nt in length. ndhF is known to be a pseudogene inuscuta (Haberhausen and Zetsche, 1994) and evi-ence suggests that it may be one in much of the rest ofonvolvulaceae also (R. Olmstead and S. Stefanovic,

ntinued
Co

tba

ttHi

102 OLMSTEAD ET AL.

unpublished). The placement of Ipomoea as sister tohe Solanaceae is consistent with published resultsased on rbcL, for which Ipomoea exhibits no ratecceleration. The Hydrostachys sequence was ex-

FIG. 2. One of the eight most-parsimonious trees based on ndhinferred nucleotide substitutions, insertions, and deletions (scale lowhollow bars indicate homoplastic insertions and deletions. (a) Outgr

remely divergent, including a region in the middle ofhe sequence that could not be aligned with confidence.owever, it did not contain excessive deletions or any

ndication of being a pseudogene. Hydrostachys also

quences drawn with branch lengths proportional to the number ofright). Solid bars indicate nonhomoplastic insertions and deletions;s, Cornales, Ericales, and Euasterids II. (b) Euasterids I.

F seer

oup


exhibits highly divergent sequences for rbcL (Hempelet al., 1994) and 18S rDNA (Albach, 1998).

One member of Caryophyllidae (Phytolacca) was in-cluded in the outgroup sampling and fell outside theAsteridae, in contrast to the results of an 18S rDNAanalysis of angiosperms, which found Caryophyllidae(including Phytolacca) to be nested within Asteridae(Soltis et al., 1997). However, finding Caryophyllidae tobe nested within Asteridae was considered anomalous

FIG. 2—

by the authors of that study (Soltis et al., 1997), whocited cpDNA studies (e.g., Chase et al., 1993) and other,unpublished studies of 18S sequences that did not findthat result. Also, combined cpDNA and 18S rDNAanalyses support the exclusion of Caryophyllidae fromAsteridae (Soltis et al., 1998).

Within Asteridae the relationships inferred fromndhF sequences are highly consistent with prior rbcLresults (e.g., Olmstead et al., 1993), but are more fully

ntinued
Co

ues for all of the more inclusive clades in the Cornales

bicassrdtnaa

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104 OLMSTEAD ET AL.

resolved and typically with greater support for cladesfound therein (Fig. 3). The classification depicted inFig. 1 and discussed below is consistent with the newlyproposed classification of the Angiosperm PhylogenyGroup (1998). Four main groups, Cornales, Ericales,Euasterids I, and Euasterids II, not previously circum-scribed in any authority-based classification (e.g.,Cronquist, 1981; Thorne, 1992), comprise the Asteri-dae. Support for the monophyly of a group composed ofthe two Euasterid groups is moderate (70% bootstrapsupport), but support for the monophyly of Euasteridswith Ericales is weak (30%), indicating that the posi-tion of Cornales as the basal-most branch is not firmlyresolved. The 18S rDNA sequences find similar groups(Soltis et al., 1997), but sampling is limited in somegroups (e.g., Cornales) and there are numerous smalldifferences in circumscription, as well as differences inrelationships, including Lamiales, that are paraphyl-etic with respect to Gentianales and Solanales and theinclusion of Caryophyllidae within Ericales.

Cornales. This clade is the smallest of the four ma-jor clades and contains families assigned by Cronquist(1981) to the Rosidae (Cornaceae, Hydrangeaceae), Dil-leniideae (Loasaceae), and Asteridae (Hydrostachya-ceae). The group is weakly supported here (31%), duein part to the very long terminal branch leading toHydrostachys. In many bootstrap samples, Hydro-stachys falls outside the Cornales. However, with Hy-drostachys removed from the analysis, bootstrap val-

FIG. 3. Reduced cladograms of rbcL (Olmstead et al., 1993) andndhF trees showing bootstrap support for the Asteridae and the 11clades recognized by Olmstead et al. (1993) within the Asteridae.Numbers in parentheses in the ndhF tree represent bootstrap valueswhen Hydrostachys is not included (Hydrostachys was not includedin the rbcL analysis). Taxon sampling in the rbcL study was differentfrom that of this study, which may account for some of the differencein resolution and magnitude of bootstrap support. Only one taxonwas included in Garryales; so, bootstrap support was not applicable.

rise dramatically (Fig. 1), as do values for major divi-sions in the Asteridae, probably due to the numerousalternative placements of Hydrostachys in variousootstrap replicates. The strength of support for thenclusion of Hydrostachys within Cornales cannot beonsidered strong on the basis of ndhF sequenceslone. However, rbcL sequences also place Hydro-tachys in this clade close to Hydrangeaceae with aimilarly long branch (Hempel et al., 1995) and 18SDNA sequences suggest a similar placement near Hy-rangeaceae (Albach, 1998). Additional molecular sys-ematic studies within this group have focused on Cor-aceae, including Nyssaceae and Alangiaceae (Xiang etl., 1993, 1998), Hydrangeaceae (Soltis et al., 1995),nd Loasaceae (Hempel et al., 1995).

Ericales. Most of the plant families belonging to thericales (sensu Angiosperm Phylogeny Group, 1998)ere assigned to subclass Dilleniidae, including all orart of the orders Diapensiales, Ebenales, Ericales, Ne-enthales, Primulales, Theales, and Violales (Cronquist,981), with a few elements from the Asteridae (e.g., Po-emoniaceae) and Rosidae (e.g., Balsaminaceae). Thislade is the most poorly represented of the four majorlades in our study. There is moderate bootstrap supportor this clade (64%), but relatively weak support for rela-ionships within the clade (Fig. 1), a pattern observed inbcL studies as well (Kron et al., 1993; Olmstead et al.,993; Morton et al., 1996, 1997). A study of EbenalesMorton et al., 1996) has shown that the traditionallyircumscribed order is polyphyletic. Molecular systematictudies of Ericaceae (Kron and Chase, 1993; Cullings,994; Kron, 1996, 1997; Kron and King, 1996), Lecyth-diaceae (Morton et al., 1997), Polemoniaceae (Steele andilgalys, 1994; Johnson and Soltis, 1995; Johnson et al.,996; Porter, 1997; and see Grant, 1998), and Sarraceni-ceae (Bayer et al., 1996) have helped resolve relation-hips within those families, but relationships among fam-lies remain poorly known.

Euasterids I. Takhtajan (1987) split his former As-eridae (Takhtajan, 1980) into two groups, Lamiidaend Asteridae, which correspond roughly to the Euas-erids I and Euasterids II (in part), respectively. Euas-erids I is supported moderately (53%), with Garryalesrepresented only by Garrya here) as a basal branchconsistent with rbcL evidence, Olmstead et al., 1993;

Chase et al., 1993; Rice et al., 1997), but the remainderf the clade, exclusive of Garryales, is more stronglyupported (75%, Fig. 1). Four main groups are identi-ed within this portion of the clade, Solanales, Boragi-aceae, Gentianales, and Lamiales, with weak supportor any of the possible relationships among them. Theentianales and Lamiales each are well supported (95nd 100%, respectively), but the Solanales, circum-cribed here to include Solanaceae, Convolvulaceae,nd a small clade represented by Montinia and Hyd-olea in this study, are weakly supported (25%). The

Boraginaceae (including Hydrophyllaceae) are well

oli

sampling with rbcL (Bremer et al., 1995). Molecular stud-


supported (97%) but weakly placed as sister to theSolanales (15%).

The most thorough previous study of the Euasterids Iclade based on rbcL was unable to resolve the placementf the Boraginaceae and recognized a separate Boragina-es (Olmstead et al., 1993). However, several other stud-es of rbcL sequences with different taxonomic sampling

have suggested a close relationship with the Solanales(e.g., Olmstead et al., 1992; Chase et al., 1993; Cosner etal., 1995), albeit weakly in all cases. The classificationadopted by the Angiosperm Phylogeny Group (1998)takes a conservative approach, leaving Boraginaceae un-assigned to an order within the Euasterids I. Support formonophyly of Solanaceae with Convolvulaceae is modest(52%), but this stems in part from the highly divergentndhF sequence of Ipomoea (Figs. 1 and 2). Support forthis clade is stronger with rbcL (Fig. 3). All prior studiesof rbcL and 18S rDNA sequences support monophyly ofSolanaceae plus Convolvulaceae (Olmstead et al., 1992,1993; Chase et al., 1993; Cosner et al., 1994; Soltis et al.,1997). Several molecular systematic studies have sub-stantially resolved relationships within Solanaceae (Olm-stead and Palmer, 1992, 1997; Spooner et al., 1993; Olm-stead and Sweere, 1994; Mione et al., 1994; Fay et al.,1998; Olmstead et al., 2000), but comparable work is lessadvanced in Convolvulaceae (S. Stefanovic and R. Olm-stead, unpublished), hampered by the difficulty in obtain-ing ndhF sequences in the family (R. Olmstead, unpub-lished). Molecular studies provide firm evidence tosupport the inclusion of Hydrophyllaceae within Boragi-naceae (Ferguson, 1998; R. Olmstead and D. Ferguson,unpublished). The clade composed of Montinia (Montini-aceae) and Hydrolea (Hydroleaceae) was first identifiedby Cosner et al. (1994) based on rbcL and includes Sphe-nocleaceae (Cosner et al., 1994) and Grevea, also in Mon-tiniaceae (B. Bremer, unpublished). Evidence from ndhF,presented here (Fig. 1) and based on rbcL (Cosner et al.,1994) provide weak support (25%) for the monophyly ofthe Montinia clade with the Solanaceae and Convolvu-laceae.

Within Gentianales, ndhF results support inclusion ofAsclepiadaceae (Araujia, Cryptostegia, Matelia) withinApocynaceae (Nerium, Thevetia) and separation ofGelsemiaceae from Loganiaceae, as indicated by rbcLstudies (Olmstead et al., 1993; Sennblad and Bremer,1996). Relationships also are congruent with those basedon rbcL (Olmstead et al., 1993; Bremer et al., 1995) infinding Rubiaceae as sister to the rest of the order and infinding poorly resolved relationships among the otherfamilies, with the exception that ndhF strongly supports(88%) a relationship between the Loganiaceae and theGentianaceae (Fig. 1). The large family Rubiaceae hasbeen subjected to several molecular systematic studies(Bremer and Jansen, 1991; Bremer, 1992, 1995; Manenet al., 1994; Bremer et al., 1995; Andreasen et al., 1998)and relationships within the family based on limitedsampling here are congruent with those based on greater

ies have resolved the placement of several genera some-times assigned to the Gentianales to other orders, includ-ing Retzia, Buddleja, Desfontainia, etc. (Bremer et al.,1994; Oxelman, et al., 1999).

The Lamiales are the largest order within the Euas-terids I, with approximately 23 families and over 20,000species. Support for the order as a monophyletic group(Fig. 1) is strong (100%), as is support for monophyly ofOleaceae (100%), which forms the sister group to the restof the clade (99%). Tetrachondraceae, a family with 1species in New Zealand and 1 in Patagonia, and Polypre-mum (monotypic, South America) are not included here,but may belong at or near the base of the order (Wagstaffand Olmstead, 1997; Oxelman et al., 1999). Gesneriaceaeform a well-supported clade (100%), with Cyrtandroideae(Cyrtandra) and Gesnerioideae (Codonanthe, Nematan-thus, Drymonia) forming sister groups, in agreementwith molecular studies of the family (Smith et al.,1997a,b). The location of Gesneriaceae near the base ofthe Lamiales is consistent with the results of other mo-lecular studies (Olmstead and Reeves, 1995; Scotland etal., 1995; Wagstaff and Olmstead, 1997; Oxelman et al.,1999). However, resolution of interfamilial relationshipsin the rest of the Lamiales is poor. Individual families arewell resolved, including Lamiaceae (99%), Acanthaceae(80%), Verbenaceae (100%), Buddlejaceae (100%), Big-noniaceae (97%), and Stilbaceae (100%). Representativesof Scrophulariaceae included here form two well-sup-ported groups, Scrophulariaceae s.s. (79%), which forms amonophyletic group (100%) with Buddlejaceae (Oxelmanet al., 1999), and Veronicaceae (97%), which includesCallitrichaceae, Hippuridaceae, and Plantaginaceae (inagreement with Olmstead and Reeves, 1995; Reeves andOlmstead, 1998). Additional work on Scrophulariaceaesuggests that a third major group, Orobanchaceae, in-cluding the achlorophylous holoparasites conventionallyassigned to the family and the green hemiparasites as-signed to Scrophulariaceae s.l., can be identified (dePam-philis et al., 1997; Wolfe et al., 1997; Nickrent et al., 1998;Young et al., 1999; Wolfe and dePamphilis, 1998; Olm-stead et al., unpublished). Molecular studies have con-tributed to the realignment of the Lamiaceae and Ver-benaceae (Cantino et al., 1992, 1997, 1998; Wagstaff etal., 1995, 1998; Wagstaff and Olmstead, 1997; Steane etal., 1997), with Verbenaceae restricted to the former sub-family Verbenoideae and with most of the rest of Verben-aceae and Symphoremaceae transferred to Lamiaceae.Molecular data support the separation of several smallfamilies (not represented here) often assigned to Verben-aceae, including Avicenniaceae, Cyclocheilaceae (Nesoge-naceae in Wagstaff and Olmstead, 1997), and Phry-maceae (Wagstaff and Olmstead, 1997). Other familiesthat have been subject to molecular systematic studyinclude Acanthaceae (Scotland et al., 1995; Hedren et al.,1995; McDade and Moody, 1999), Bignoniaceae (Span-gler and Olmstead, 1999), and Oleaceae (Kim andJansen, 1998; Wallander and Albert, 1998).

Euasterids II. The second Euasterid clade (Fig. 1)aw(Ea(ic

sAaib

aea1sBJnaCwroosp(Cca1aa(a

sCDlalSoVss1a

With limited sampling in the two large families Api-alsbaiarwAscfiss1

fmatr(HMMpstqct

pqstpAGrJriiml

106 OLMSTEAD ET AL.

lso is supported moderately (68%) and comprises fourell-supported orders: Apiales (100%), Aquifoliales

100%), Asterales (99%), and Dipsacales (83%). Unlikeuasterids I, relationships among orders in this cladere clear, with Apiales and Dipsacales forming a clade65%), which is sister to Asterales (90% support for thenclusive clade), and Aquifoliales being sister to thelade comprising the other three orders (Fig. 1).

Takhtajan’s (1987) Asteridae sensu stricto corre-ponds very closely to the group recognized as thesterales here and includes the Asterales, Calycerales,nd Campanulales of Cronquist (1981). Relationshipsnferred within this clade correspond closely to thoseased on rbcL studies, with a few exceptions. Most

studies based on rbcL supported monophyly of Calyc-eraceae with Goodeniaceae, which together form thesister group to Asteraceae (Olmstead et al., 1993;Michaels et al., 1993; Cosner et al., 1995; Gustafsson et

l., 1996). However, in this study, monophyly of Calyc-raceae with Asteraceae is strongly supported (98%)nd the family Goodeniaceae is sister to that clade (Fig.). This also was found, but with less support, in thetudies of Kim and Jansen (1995), based on ndhF,acklund and Bremer (1997), based on rbcL, andansen and Kim (1996), based on combined rbcL anddhF sequences. This broad concept of Asterales isccepted here, because segregating a narrowly definedampanulales (Campanulaceae plus Stylidiaceae)ould leave a poorly supported clade (37%), and seg-

egating a narrowly defined Asterales (Asteraceaenly) would leave a paraphyletic grade of small groupsften comprising individual families. All families withampling of two or more species were strongly sup-orted monophyletic groups, including Asteraceae80%), Goodeniaceae (97%), Menyanthaceae (95%), andampanulaceae (100%). Asteraceae have been the fo-

us of extensive molecular systematic study (Jansennd Palmer, 1987; Jansen et al., 1991, 1992; Kim et al.,992; Kim and Jansen, 1995; Jansen and Kim, 1996)nd other studies have focused on the relationshipsmong families most closely related to AsteraceaeMichaels et al., 1993; Gustafsson et al., 1996; Jansennd Kim, 1996; Backlund and Bremer, 1997).Within Dipsacales, ndhF results concur with rbcL

tudies (Donoghue et al., 1992; Olmstead et al., 1993;hase et al., 1993; Backlund and Bremer, 1997; M.onoghue, R. Olmstead, and T. Eriksson, unpub-

ished), which suggest that the traditional Caprifoli-ceae are paraphyletic with respect to the other fami-ies in the order (Figs. 1 and 2). Adoxaceae (includingambucus and Viburnum) are sister to the rest of therder. The Caprifoliaceae without Sambucus andiburnum still are paraphyletic with respect to Dip-acaceae and Valerianaceae. Molecular phylogenetictudies have examined Adoxaceae (Baldwin et al.,995; Eriksson and Donoghue, 1997) and Valeri-naceae (Backlund and Bremer, 1997).

ceae and Araliaceae, which make up most of the Apia-es, each appears to be monophyletic (Fig. 1). However,ubfamily Hydrocotyloideae (Apiaceae), which haseen found to be basal and paraphyletic (Plunkett etl., 1996, 1997; Downie et al., 1998), was not sampledn this study. Our results suggest that Pittosporaceaend Apiaceae form a clade (81%), in contrast to somebcL studies (Chase et al., 1993; Plunkett et al., 1996),hich find Pittosporaceae to be sister to a combinedpiaceae/Araliaceae, though with weak support. Othertudies of rbcL (Olmstead et al., 1993) and matK andombined matK/rbcL (Plunkett et al., 1997) data setsnd that this relationship is unresolved. The largeubfamily Apioideae has been the subject of additionaltudy with both nuclear (Downie and Katz-Downie,996) and chloroplast (Downie et al., 1996) DNA.The Aquifoliales comprise a group of three small

amilies, Aquifoliaceae, Helwingiaceae, and Phyllono-aceae. In this study, Aquifoliaceae and Helwingi-

ceae are strongly supported (92%), whereas both al-ernate relationships have been suggested by differentbcL studies, albeit with lower bootstrap supportAquifoliaceae/Phyllonomaceae—Olmstead et al., 1993;elwingiaceae/Phyllonomaceae—Chase et al., 1993;organ and Soltis, 1993; Backlund and Bremer, 1997).organ and Soltis (1993) note a shared leaf venation

attern in Helwingia and Phyllonoma, in addition to thehared characteristic of epiphyllous inflorescences. Thesewo taxa also share a 9-bp deletion in their ndhF se-uence, which was included in this analysis as a binaryharacter weighted equally with nucleotide substitu-ions.

With minor exceptions, the ndhF results confirmrevious hypotheses of relationship based on rbcL se-uences and provide greater resolution and increasedupport for those hypotheses. The congruence betweenhe phylogenetic results from these two gene sequencesrovides a foundation for a revised classification ofsteridae at the ordinal level (Angiosperm Phylogenyroup, 1998). The greater ability of ndhF sequences to

esolve intrafamilial relationships (e.g., Kim andansen, 1995; Olmstead and Reeves, 1995), relative tobcL, means that data from studies of individual fam-lies based on ndhF may be integrated more effectivelynto a broad-scale study of Asteridae phylogeny and

ay ultimately help to resolve the remaining family-evel problem areas (e.g., Lamiales, Ericales).

ACKNOWLEDGMENTS

This research has been supported by NSF Grant BSR-9107827 toR.G.O. and Grant DEB-9020171 to R.K.J. Thanks to P. Reeves andtwo anonymous reviewers for helpful comments. Thanks to the manyindividuals and Botanical Gardens who helped by providing planttissue or DNA for analysis.

APPENDIX 1

List of Taxa Included in Study, Sources of Plant Material, and GenBank Accession Nos.

Order/family Species DNA source/voucherGenBank

Accession No.

OutgroupsSapindaceae aAcer negundo L. K.-J. Kim 13785 (YNUH) AF130227Ceratophyllaceae aCeratophyllum demersum L. K.-J. Kim 13602 (TEX) AF130232Cercidiphyllaceae aCercidiphyllum japonicum Siebold & Zucc. AA-23185A; I. Hay, M.

Harrison, & S. Kulik 4061(A)

AF130224

Fagaceae aQuercus rubra L. UC Campus, no voucher AF130226Magnoliaceae aLiriodendron tulipifera L. K.-J. Kim 13743 (YNUH) AF130230Magnoliaceae aMagnolia sieboldii Koch K.-J. Kim 13754 (YNUH) AF130231Paeoniaceae aPaeonia tenuifolia L. Kron 2115 AF130223Papaveraceae aDicentra spectabilis (L.) Lem. K.-J. Kim 13700 (YNUH) AF130234Phytolaccaceae aPhytolacca americana L. K.-J. Kim 13766 (YNUH) AF130229Ranunculaceae aAquilegia bicolor Ehrh. UC Campus, no voucher AF130233Rhamnaceae aRhamnus davurica Pall. K.-J. Kim 13840 (YNUH) AF130225Saxifragaceae aHeuchera micrantha Dougl. ex Lindl. Soltis and Soltis 1949 WS AF130222Staphyleaceae aStaphylea emodi Wall. AA-478-78; no voucher AF130228

CornalesCornaceae aAlangium platanifolium Harms K.-J. Kim 13748 (YNUH) AF130221Cornaceae aCornus florida L. K.-J. Kim 13693 (YNUH) AF130220Hydrangeaceae aCarpenteria californica Torrey Soltis and Soltis 2478 WS AF130217Hydrangeaceae aHydrangea macrophylla Ser. K.-J. Kim 13777 (YNUH) AF130218Hydrostachyaceae bHydrostachys multifida A. Juss. G. Schatz et al. 3413 (MO) AF147712Loasaceae aEucnide rupestris (Baill.) Thomps. & Ernst Powell 1707 (TEX) AF130219

EricalesBalsaminaceae aImpatiens capensis Meerb. Chase 114 AF130210Ebenaceae aDiospyros texana Scheele K.-J. Kim 12776 (TEX) AF130213Ericaceae aRhododendron mucronulatum Turcz. K.-J. Kim 13736 (YNUH) AF130209Polemoniaceae aPhlox drummondii Hook. K.-J. Kim 12898 (TEX) AF130211Primulaceae aAnagallis arvensis L. K.-J. Kim 12894 (TEX) AF130212Styracaceae aHalesia carolina L. AA-22308A I, Hay & M.

Gilmore 4 (A)AF130214

Styracaceae aStyrax americana Lam. UC Campus, no voucher AF130215Theaceae aCamellia japonica L. K.-J. Kim 13593 (YNUH) AF130216

Euasterids IGarryales/Garryaceae bGarrya elliptica Dougl. ex Lindl. Rancho Santa Ana B.G.; no

voucherAF147714

Gentianales/Apocynaceae aAraujia sericifera Brot. UC GH 850807, no voucher AF130165Gentianales/Apocynaceae aCryptostegia grandiflora R. Br. UC GH, no voucher AF130167Gentianales/Apocynaceae aMatelea biflora (Rafin.) Woods. K.-J. Kim 12897 (TEX) AF130166Gentianales/Apocynaceae aNerium oleander L. K.-J. Kim 12809 (TEX) AF130168Gentianales/Apocynaceae aThevetia peruviana K. Schum. J. Panero s.n. (TEX) AF130169Gentianales/Gelsemiaceae aGelsemium sempervirens (L.) Ait. K.-J. Kim 12807 (TEX) AF130170Gentianales/Gentianaceae bExacum affine Balf. f. ex Regel Matthaei B. G., Ann Arbor, MI;

no voucherAF147710

andAF147711

Gentianales/Gentianaceae bGentiana procera Holm H. Michaels, coll., Westland,MI; no voucher

L36400

Gentianales/Loganiaceae bSpigelia marilandica L. Beal B. G., East Lansing, MI;no voucher

AF147713

Gentianales/Rubiaceae aCephalanthus occidentalis L. K.-J. Kim 12498 (TEX) AF130173Gentianales/Rubiaceae aErithalis fruticosa Urb. Meagher 990 (FTG) AF130174Gentianales/Rubiaceae aGuettarda uruguensis Cham. & Schltdl. Gillis 9575 (FTG) AF130172Gentianales/Rubiaceae aIxora parviflora Vahl. Bremer 3104 (UPS) AF130176Gentianales/Rubiaceae aPentas lanceolata (Forssk.) Deflers UC GH 850651, no voucher AF130175Gentianales/Rubiaceae aRogiera suffrutescens (Brandegee) Borhidi Bremer 2712 (S) AF130171Lamiales/Acanthaceae aJusticia carnea Lindl. UC GH 850161, no voucher AF130155Lamiales/Acanthaceae bThunbergia alata Bojer R. Olmstead 93-45 (WTU) U12667Lamiales/Veronicaceae aDigitalis purpurea L. K.-J. Kim 13943 (YNUH) AF130150Lamiales/Veronicaceae aPlantago lanceolata L. K.-J. Kim 13790 (YNUH) AF130151Lamiales/Veronicaceae bCallitriche hermaphroditica L. T. Philbrick 3022 (CONN) L36441



Accession No.

Lamiales/Veronicaceae bVeronica catenata Pennell R. Olmstead 92-144 (WTU) L36419Lamiales/Bignoniaceae aCampsis radicans Seem. K.-J. Kim 12802 (TEX) AF130144Lamiales/Bignoniaceae aTecoma stans Juss. K.-J. Kim 12805 (TEX) AF130145Lamiales/Bignoniaceae bCatalpa speciosa Warder R. Olmstead 88-003 (WTU) LL36397Lamiales/Buddlejaceae aBuddleja davidii Franchet Cult. in NYBG, s.n.; no voucher AF130143Lamiales/Buddlejaceae bNicodemia diversifolia (Vahl) Tenore B. Bremer 3099 (UPS) L36405Lamiales/Gesneriaceae aCodonanthe digna Wiehler UC GH 920093, no voucher AF130156Lamiales/Gesneriaceae aCyrtandra hawaiensis C. B. Clarke Cult. in Hawaii Tropical

Garden, Manoa; no voucherAF130159

Lamiales/Gesneriaceae aDrymonia strigosa (Oerst.) Wiehler UC GH, no voucher AF130158Lamiales/Gesneriaceae aNematanthus ‘Butterscotch’ (CV. of N. longipes DC.) UC GH 850103, no voucher AF130157Lamiales/Lamiaceae aClerodendrum trichotomum Thunb. B.Sun, s.n. (CNUH) AF130146Lamiales/Lamiaceae aTetraclea coulteri A. Gray K.-J. Kim 10026 (TEX) U78706Lamiales/Lamiaceae aVitex agnus-castus L. K.-J. Kim 2804 (TEX) U78707Lamiales/Lamiaceae bAjuga reptans L. S. J. Wagstaff 89-07 (BHO) L36391Lamiales/Lamiaceae bMentha rotundifolia (L.) Huds. S. J. Wagstaff 89-026 (BHO) U78696Lamiales/Myoporaceae bMyoporum mauritianum A. DC. R.B.G. Kew No. 1984-4220; no

voucherL36403

Lamiales/Oleaceae aJasminum mesnyi Hance K.-J. Kim 12810(TEX) AF130162Lamiales/Oleaceae aLigustrum vulgare L. AA 486-88; no voucher AF130164Lamiales/Oleaceae aNyctanthes arbor-tristis L. Y. Dave s.n. (Sardar Patel

Univ. Herbarium, India)AF130161

Lamiales/Oleaceae aOlea europeaa L. UC GH 850974, no voucher AF130163Lamiales/Paulowniaceae bPaulownia tomentosa Steud. R. Olmstead 88-008 (WTU) L36406Lamiales/Pedaliaceae bSesamum indicum L. Cult.; no voucher L36413Lamiales/Scrophulariaceae bScrophularia californica Cham. & Schldl. C. W. dePamphilis s.n. L36411Lamiales/Scrophulariaceae bSelago thunbergii Choisy C. W. dePamphilis s.n. L36412Lamiales/Scrophulariaceae bVerbascum thapsus L. R. Olmstead, coll., Boulder, CO

no voucherL36417

Lamiales/Stilbaceae bEuthystachys abbreviata (E. Mey) A. DC. McDonald & Rourke, coll., S.Africa; no voucher

AF147715

Lamiales/Stilbaceae bRetzia capensis Thunb. Kallersjo 0401191 (BOL) AF147716Lamiales/Verbenaceae aAloysia gratissima (Gill & Hook.) Tronc. K.-J. Kim 12803 (TEX) AF130154Lamiales/Verbenaceae aLantana horrida H.B.K. K.-J. Kim 12808 (TEX) AF130152Lamiales/Verbenaceae aPhyla incisa Small K.-J. Kim 12801 (TEX) AF130153Solanales/Convolvulaceae aIpomoea batata Lam. K.-J. Kim 13844 (YNUH) AF130177Solanales/Hydroleaceae bHydrolea ovata Nutt. ex Choisy R. Olmstead 89-009 (WTU) AF013999Solanales/Montiniaceae aMontinia caryophyllacea Thunb. Williams 2833 (MO) AF130178Solanales/Solanaceae bNicotiana tabacum L. Matthaei B.G., Ann Arbor, MI;

no voucherL14953

Solanales/Solanaceae bNolana spathulata Ruiz & Pav. M. O. Dillon 3767 (F) U08925Solanales/Solanaceae bPetunia axillaris (Lam.) B.S.P. Seed BIRM/s.0367; RGO S-60

(WTU)U08926

Solanales/Solanaceae bSchizanthus pinnatus Ruiz & Pav. Seed BIRM/s.0224; RGO S-72(WTU)

U08929

Solanales/Solanaceae bSolanum lycopersicum L. Cult.; no voucher U08921Unassigned/Boraginaceae aPhacelia patuliflora (Engln. & Gray) Gray K.-J.Kim 12899 (TEX) AF130179Unassigned/Boraginaceae bBorago officinalis L. Matthaei B.G., Ann Arbor, MI;

no voucherL36393

Unassigned/Boraginaceae bHydrophyllum virginiana L. R. Olmstead, coll., Ann Arbor,MI; no voucher

AF019646

Euasterids IIApiales/Apiaceae aAngelica gigas Nakai K.-J. Kim 13948 (YNUH) AF130198Apiales/Apiaceae aCoriandrum sativum L. UC Pharmacy Garden, no

voucherAF130199

Apiales/Apiaceae aOsmorhiza claytoni (Michx.) C. B. Clarke J. Wen 850 (GH) AF130200Apiales/Araliaceae aAcanthopanax sessiliflorus Seem. K.-J. Kim 13557 (YNUH) AF130202Apiales/Araliaceae aHedera helix L. K.-J. Kim 13594 (YNUH) AF130203Apiales/Araliaceae aPanax ginseng C. Meyer K.-J. Kim 13555 (CNUH) AF130204Apiales/Griseliniaceae aGriselinia lucida G. Forst. Cameron s.n. (AUK) AF130205Apiales/Pittosporaceae aPittosporum tobirum Ait. K.-J. Kim 13594 (YNUH) AF130201Aquifoliales/Aquifoliaceae aIlex crenata Thunb. K.-J. Kim 12811 (TEX) AF130206Aquifoliales/Helwingiaceae aHelwingia japonica (Thunb.) F. Dietr. AA-912; I. Hay & M. Harrison

5579 (A)AF130207

108 OLMSTEAD ET AL.

APPENDIX 1—Continued

REFERENCES

B

B

B

sification and comparative ecology. Ann. Mo. Bot. Gard. 79: 380–387.

B

B

C


Accession No.

Aquifoliales/Phyllonomaceae aPhyllonoma laticuspis (Turcz.) Engl. D. Morgan 2124 (WS) AF130208Asterales/Argophyllaceae aCorokia cotoneaster Raoul Strybing Arb. 74-211; no

voucherAF130182

Asterales/Asteraceae aAster cordifolius L. R. Jansen 906 (MICH) L39449Asterales/Asteraceae aBarnadesia caryophylla (Vell.) Blake Kew 001-76-0038; no voucher L39394Asterales/Asteraceae aDasyphyllum argenteum Kunth Stuessy & Viteri 12464 (OS) L39392Asterales/Asteraceae aGerbera jamesonii Bolus R. Jansen 915 (MICH) L39403Asterales/Asteraceae aHelianthus annuus L. Price Ha89 (TAES) L39382Asterales/Asteraceae aLactuca sativa L. Cult.; no voucher L39389Asterales/Calyceraceae aBoopis anthemoides Juss. Hunziker 25258 (CORD) L39304Asterales/Campanulaceae aCampanula ramulosa Wall. R. Jansen 984 (MICH) L39387Asterales/Campanulaceae aCodonopsis lanceolata Trautv. K.-J. Kim 13942 (YNUH) AF130185Asterales/Campanulaceae aCyananthus microphyllus Benth. M. Cosner 176 (OS) AF130186Asterales/Campanulaceae aLobelia cardinalis L. UC GH 980112; no voucher AF130187Asterales/Campanulaceae aTrachelium caeruleum L. M. Cosner 173 (OS) AF130184Asterales/Goodeniaceae aDampiera diversifolia De Vriese Kew 420-84-04494; no voucher L39386Asterales/Goodeniaceae aScaevola frutescens Krause MBG 19426; no voucher L39385Asterales/Menyanthaceae aMenyanthes trifoliata L. Cult. in NYBG, s.n.; no voucher L39388Asterales/Menyanthaceae aNymphoides indica (L.) O.Ktze. Cult. in NYBG s.n.; no voucher AF130181Asterales/Menyanthaceae aVillarsia calthifolia F.Muell. R. Ornduff 9726 (UC) AF130180Asterales/Pentaphragmataceae aPentaphragma elliptica Poulsen Hugh Tan s.n. (Natl. Univ. of

Singapore)AF130183

Asterales/Stylidiaceae aStylidium graminifolium Sw. M. Cosner 174 (OS) AF130188Dipsacales/Adoxaceae aSambucus canadensis L. K.-J. Kim 12887 (TEX) AF130196Dipsacales/Adoxaceae aViburnum sargentii Koehne K.-J. Kim 13768 (YNUH) AF130197Dipsacales/Caprifoliaceae aLonicera japonica Thunb. K.-J. Kim 13767 (YNUH) AF130194Dipsacales/Caprifoliaceae aSymphoricarpos orbiculatus Moench G.Nesom 7741 (TEX) AF130195Dipsacales/Dipsacaceae aDipsacus sativus (L.) Honck. R. Jansen 931 (MICH) AF130190Dipsacales/Dipsacaceae aScabiosa mansenensis Nakai K.-J. Kim 13889 (YNUH) AF130189Dipsacales/Dipsacaceae aSuccisa pratensis L. UC Pharmacy Garden, no

voucherAF130191

Dipsacales/Linnaeaceae aAbelia x grandiflora (Andre) Rehder K.-J. Kim 12841 (TEX) AF130193Dipsacales/Valerianaceae aValeriana fauriei Briq. K.-J. Kim 13839 (YNUH) AF130192

Note. AA, Arnold Arobretum; NYBG, New York Bot. Gard.; UC, University of Connecticut. Classification follows Angiosperm PhylogenyGroup (1998).

a Sequences determined in Jansen lab.b Sequences determined in Olmstead lab.


APPENDIX 1—Continued

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