Copyright © NISC Pty Ltd South African Journal of Botany 2004, 70(3): 393–406 Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299 Circumscription of Apiaceae tribe Oenantheae TM Hardway1, K Spalik2, MF Watson3, DS Katz-Downie1 and SR Downie1* 1 Department of Plant Biology, University of Illinois, Urbana 61801, United States of America Department of Plant Systematics and Geography, Warsaw University, Aleje Ujazdowskie 4, 00-478 Warsaw, Poland 3 Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, United Kingdom * Corresponding author, email: sdownie@life.uiuc.edu 2 Received 18 August 2003, accepted in revised form 17 November 2003 Previous molecular systematic investigations into the higher-level relationships of Apiaceae subfamily Apioideae have revealed a strongly supported clade recognised as tribe Oenantheae Dumort. These plants may have clusters of fibrous or tuberous-thickened roots, corky-thickened fruits, and other adaptations for existence in wet or aquatic habitats. In some species, the leaves may be finely dissected or linear-septate and much reduced. We have initiated collaborative studies to produce a comprehensive estimate of phylogeny of the tribe, but such investigations are thwarted because information on the composition of the tribe is lacking. Herein, tribe Oenantheae is circumscribed to include the following genera: Afrocarum, Berula, Bifora (pro parte), Cicuta, Cryptotaenia (pro parte), Cynosciadium, Daucosma, Helosciadium, Lilaeopsis, Limnosciadium, Neogoezia, Oenanthe, Oxypolis, Perideridia, Ptilimnium, Sium, and Trepocarpus. Relationships inferred from phylogenetic analyses of nuclear rDNA ITS sequences from 64 accessions representing all 17 genera reveal that four genera are not monophyletic. Bifora and Cryptotaenia have members that fall outside of the tribe; Berula and Sium each comprise two or more lineages within Oenantheae. The St Helena endemics, Sium bracteatum and S. burchellii, ally with African Berula erecta; this clade is sister to the African endemic species Sium repandum and Afrocarum imbricatum, and this entire group is allied closely with north temperate Berula erecta. Nomenclatural changes are in order, but must await further study. Representatives of eight genera native to North America comprise a monophyletic group, and results from relative rate tests suggest that this lineage is evolving much faster than any other major clade recognised within the tribe. Introduction In all higher-level molecular systematic studies of Apiaceae subfamily Apioideae to date, whether based on chloroplast DNA (cpDNA) restriction site comparisons or sequences from the nuclear ribosomal DNA internal transcribed spacer (ITS) region or several chloroplast loci, the ‘Oenanthe clade’ is revealed as a strongly supported monophyletic group (e.g. Plunkett et al. 1996, Downie et al. 1998, 2000b, Plunkett and Downie 1999). A core group of genera is recognised in many of these studies, and includes Berula W.D.J. Koch, Cicuta L., Cryptotaenia DC., Helosciadium W.D.J. Koch, Oenanthe L., Oxypolis Raf., Perideridia Rchb., and Sium L. The distinctive genera Neogeozia Hemsl. and Lilaeopsis Greene, whose affinities until recently were obscure, also belong within this clade (Plunkett et al. 1996, Petersen et al. 2002). The genera Cynosciadium DC., Limnosciadium Mathias and Constance (1944–1945), and Ptilimnium Raf. may be included as well, based on their similar vegetative and (or) fruit morphologies (Downie et al. 2000b, 2001). Thirteen genera have been recognised unequivocally as belonging to the Oenanthe clade. The Oenanthe clade can be defined morphologically, although there are no characters that are common and unique to the group. Its members are characterised generally by having fascicled roots (which may be thickened and bear tubers) and glabrous leaves and stems. They are often associated with moist to wet habitats, and some are true aquatics. Many species, including the members of Berula, Neogoezia, Perideridia, and Sium, have pinnate leaves with primary divisions that are not further divided and are often rounded and of equal size, superficially resembling those leaves of members of the distantly related genus Pimpinella L. Species inhabiting moist to wet habitats, including representatives of Helosciadium, Sium, and Oenanthe, often have submerged leaves that are more finely dissected and have narrower lobes than those of the air-borne ones. Leaves, particularly the petioles, are sometimes succulent and the leaf lobes are reduced. In Berula, the lowest pair of primary divisions is absent, whereas in Lilaeopsis and some species of Oxypolis and Ptilimnium, all divisions are lacking and their initial number may only be inferred from the nodes (septae) extant on the leaf axis. The fruits of many members are globose to broadly-ovate and are commonly 394 Hardway, Spalik, Watson, Katz-Downie and Downie spongy/corky thickened, facilitating dispersal in water (Affolter 1985, Wilm and Taft 1998). Indeed, Darwin (1859) was impressed by the lengthy flotation and subsequent germination abilities of Helosciadium. Lilaeopsis and Neogoezia have simple umbels, but this is an extremely rare feature of Apiaceae subfamily Apioideae. However, none of these attributes can be considered a synapomorphy for the clade, as each of these features can be found in genera outside of the group (Petersen et al. 2002). This is not surprising, given the fact that many tribes and clades recognised in subfamily Apioideae on the basis of molecular data cannot be delimited unambiguously using morphological or anatomical data (Downie et al. 2001). No prior taxonomic treatment has grouped together those genera that are included here in the Oenanthe clade. In the system of classification of Pimenov and Leonov (1993), modified from Drude (1898), the 13 genera recognised in the group are distributed among three tribes (Apieae, Peucedaneae Dumort., and Smyrnieae Spreng.). Downie et al. (2000b) recognised this group as tribe Oenantheae Dumort., but stressed that their circumscription of the tribe is radically different from that proposed by Dumortier (1827) and others, such as Koso-Poljansky (1916) and Cerceau-Larrival (1962). In this paper, we summarise the results of our efforts to identify additional members of Apiaceae tribe Oenantheae. We then report results of phylogenetic analyses of representatives of all genera comprising the tribe, placing emphasis on its African members, specifically the genera Berula, Sium, and a new addition to the tribe, Afrocarum Rauschert. Sequences of the ITS region were employed because they have been shown to be appropriate to assess evolutionary relationships within Apiaceae subfamily Apioideae (Downie et al. 2001). Given that no previous classification has grouped together those genera here recognised as constituting tribe Oenantheae, the results of this paper should facilitate further systematic activity on this widely distributed and strongly supported monophyletic group of Apiaceae. Material and Methods Taxonomic sampling To ascertain the limits of Apiaceae tribe Oenantheae, ITS sequence data were procured from 100 accessions of subfamily Apioideae using standard PCR and sequencing methods (described below). These accessions included representation of the 13 genera unambiguously placed in the tribe as a result of earlier studies, plus Bifora americana (DC.) Benth. & Hook., provisionally included in the tribe on the basis of matK sequence comparisons (Plunkett et al. 1996). These accessions also included representation of 19 additional genera (those listed in Table 1, plus Afrocarum, Daucosma Engelm. & A. Gray ex A. Gray, and Trepocarpus Nutt. ex DC.) whose morphologies (or previous taxonomic placements, as indicated by their synonymies) suggested possible close affinities with those taxa already included in the tribe. Sixty-four accessions were considered in the phylogenetic analysis (Table 2). The phylogenetic placement of the African genus Afrocarum was ascertained by sampling putatively allied genera Berula and Sium (Cannon 1978), from Africa and elsewhere. Sampling of the genus Helosciadium was comprehensive (Wolff 1927, Z. Popper and M. Watson, unpubl. data). For Lilaeopsis and the seven exclusively North American taxa (i.e. Bifora americana, Cynosciadium, Daucosma, Limnosciadium, Neogeozia, Ptilimnium and Trepocarpus), only single exemplars of each genus were included, as their phylogenetic relationships are the subject of an ongoing, concurrent study (S. Downie et al., unpubl. data). Sampling of the remaining genera (Cicuta, Cryptotaenia, Oenanthe, Oxypolis and Perideridia) was based primarily on material availability. With the exception of Lilaeopsis, the nomenclatural type of each genus was included in this study. Table 1: Taxa examined for inclusion in Apiaceae tribe Oenantheae but excluded on the basis of phylogenetic analysis of molecular data Genus Chamaele Miq. Crenosciadium Boiss. & Heldr. ex Boiss. Cyclospermum Lag. Huanaca Cav. Lichtensteinia Cham. & Schltdl. pro parte Muretia Boiss. Naufraga Constance & Cannon Niphogeton Schltdl. Oreomyrrhis Endl. Oreoschimperella Rauschert Pimpinella L. pro parte Pterygopleurum Kitag. Pternopetalum Franch. Sclerosciadium W.D.J. Koch ex DC. pro parte Spuriopimpinella (H. Boissieu) Kitag. Stoibrax Raf. Species examined (and synonyms) C. decumbens (Thunb.) Makino (=Sium decumbens Thunb.; Oenanthe decumbens (Thunb.) Koso-Pol.) C. siifolium Boiss. & Heldr. ex Boiss. C. leptophyllum (Pers.) Sprague ex Britton & P. Wilson H. andina Phil. L. interrupta (Thunb.) Sond. (=Oenanthe interrupta Thunb.) M. lutea (M. Bieb.) Boiss. (=Sium luteum (M. Bieb.) Spreng.) N. balearica Constance & Cannon N. ternata (Willd. ex Schltdl.) Mathias & Constance (=Apium montanum Kunth) O. andicola (Kunth) Hook. f. O. verrucosa (J. Gay ex A. Rich.) Rauschert (=Sium verrucosum J. Gay ex A. Rich.) P. siifolia Leresche (=Apium siifolium (Leresche) Calest.) P. diversifolia DC. (=Helosciadium pubescens DC.) P. neurophyllum (Maxim.) Kitag. (=Perideridia neurophylla (Maxim) T.I. Chuang & Constance; Sium neurophyllum (Maxim.) Hara) P. vulgare (Dunn) Hand.-Mazz. S. nodiflorum (Schousb.) Coss. (=Oenanthe nodiflora Schousb.) S. calycina (Maxim.) Kitag. S. dichotomum (L.) Raf. (=Apium dichotomum (L.) Calest.) DNA No. or Accession Taxon Identifier 132 Afrocarum imbricatum (Schinz) Rauschert 1444 Afrocarum imbricatum 82 Berula erecta (Huds.) Coville 116 Berula erecta 150 Berula erecta 251 Berula erecta 2253 Berula erecta 2257 Berula erecta 799 Berula erecta 1467 Bifora americana Benth. & Hook. f. ex S. Watson 1923 Cicuta bulbifera L. 1935 Cicuta bulbifera 1113 Cicuta maculata L. var. angustifolia Hook. 75 Cicuta virosa L. 131 Cicuta virosa 426 Cicuta virosa 817 Cryptotaenia canadensis (L.) DC. 574 Cryptotaenia japonica Hassk. 402 1063 2397 62449 170 64358 919 15108 1871 1870 1999 2000 Tanzania, Iringa, Mufindi District, Igowole, Kayombo & Kayombo 217 (MO) AY360228 Malawi, Northern Province, Mzimba District, Mzuzu, Katoto, Pawek 5442 (MO) Zimbabwe, Alpes Rd near Harare, Bayliss 10592 (MO) Ethiopia, 90km S of Addis Ababa, Ash 859 (MO) Germany; cult. UIUC from seeds obtained from Univ. Oldenburg Bot. Gard., Downie 150 (ILL) France; cult. UIUC from seeds obtained from Conservatoire et Jardins botaniques de Nancy, Downie 251 (ILL) Denmark, Sjælland, Sorø Sønderskov, Petersen GPL28 Denmark, Sjælland, near Tuel å, Seberg OSA 486 Ethiopia; cult. Univ. California Bot. Gard., Berkeley, L. Constance pers. coll. C-2453 (UC) USA, Texas, Williamson Co., S of Jarrell, Nesom & Grimes 6415 (MO) AY360229 AY353975 (ITS-1 only) AY353976 (ITS-1 only) U79607 U79605 AY360230 AY360231 U78369 AY360232 USA, Illinois, Will Co., Keepatau Forest Preserve, Taft 794 (ILLS) Canada, Ontario, Rushing River Provincial Park, E of Kenora, Blanz & Jones 4750 (ILL) USA, Wyoming, Goshen Co., Bear Creek, Nelson et al. 33517 (RM) AY360233 AY360234 AY360235 Finland; cult. UIUC from seeds obtained from Univ. Joensuu Bot. Gard., Downie 75 (ILL) Germany; cult. UIUC from seeds obtained from Univ. Oldenburg Bot. Gard., Downie 131 (ILL) China, Yunnan, Xiao Zhongdian, RBGE Gyalthang Expedition, FED 426 (E) USA, Illinois, Champaign Co., Urbana, Downie 817 (ILL) Japan, Honshu Island, Koyosan area, McNamara et al. 90 (UC); cult. Univ. California Bot. Gard., Berkeley (no. 90.0891) China; cult. UIUC from seeds obtained from Shanghai Bot. Gard., Downie 402 (ILL) USA, Illinois, Jackson Co., Shawnee National Forest, Phillippe 21886 (ILLS) USA, Texas, Kerr Co., Kerville, Heller 1943 (MO) Spain, Balearic Islands, ex cult. Ville de Conservatoire botanique Mulhouse, France (E); cult RBGE (no. 19962449) France, Corse, Musella; cult. Conservatoire botanique de la Ville de Mulhouse (no. 2048A), Herb. Reduron s.n. U78372 U78372 AY353978, AY353985 U79613 U78367 Sicily, Messina, Davis & Sutton 64358 (E) AF164822, AF164847 Jordan, Wadi Al-Yabis, along Jordan River, Lahham & El-Oqlah 18 (Yarmouk Univ. Herb.) AF164823 Spain, Grenada, Charpin et al. AC15108 (E) France, Vaucluse, Malaucène, Herb Reduron s.n. France, Haut-Rhin, Luewenheim; cult. Conservatoire botanique de la Ville de Mulhouse (no. 9463), Herb. Reduron s.n. USA, Oregon, Douglas Co., East Gardiner, Hill & Dutton 32982 (ILLS) AY353980, AY353987 AY360240 AY360241 USA, Illinois, Champaign Co., Champaign, Hill 30580 (ILLS) AY360243 AY360236 AY360237 AY360238 AY353979, AY353986 AY360239 AY360242 395 Cryptotaenia japonica Cynosciadium digitatum DC. Daucosma laciniata Engelm. & A. Gray Helosciadium bermejoi (L. Llorens) Popper & M.F. Watson* Helosciadium crassipes W.D.J. Koch ex Rchb. Helosciadium inundatum (L.) W.D.J. Koch Helosciadium nodiflorum (L.) W.D.J. Koch Helosciadium nodiflorum Helosciadium nodiflorum Helosciadium repens (Jacq.) W.D.J. Koch Lilaeopsis occidentalis J.M. Coult. & Rose Limnosciadium pinnatum (DC.) Mathias & Constance GenBank No. Voucher Information South African Journal of Botany 2004, 70: 393–406 Table 2: Plant accessions from which nuclear ribosomal DNA ITS sequences were obtained, with corresponding voucher information and GenBank reference numbers. Two GenBank numbers per accession indicate separate ITS-1 and ITS-2 sequences (with no intervening 5.8S data); a single GenBank number (unless otherwise indicated) indicates a contiguous ITS-1, 5.8S, and ITS-2 sequence. Herbarium acronyms are according to Holmgren et al. (1990) 396 Table 2 cont. DNA No. or Accession Taxon Identifier 2138 476 Neogoezia minor Hemsl. Oenanthe banatica Heuff. 40 247 1300 165 1282 273 29 Oenanthe Oenanthe Oenanthe Oenanthe Oenanthe Oenanthe Oenanthe 521 915 1142 Oenanthe sarmentosa J. Presl ex DC. Oxypolis fendleri (A. Gray) A. Heller Oxypolis occidentalis J.M. Coult. & Rose Oxypolis rigidior (L.) J.M. Coult. & Rose Perideridia americana (Nutt. ex DC.) Rchb. Perideridia howellii (J.M. Coult. & Rose) Mathias Perideridia kelloggii (A. Gray) Mathias Perideridia kelloggii 1927 1938 780 855 635 778 2165 Perideridia kelloggii Ptilimnium capillaceum (Michx.) Raf. Sium bracteatum (Roxb.) Cronk Sium bracteatum Sium burchellii (Hook. f.) Hemsl. Sium frigidum Hand.-Mazz. Sium latifolium L. Sium latifolium Sium latifolium Sium repandum Welw. ex Hiern Sium sisarum L. Sium sisarum Sium sisarum Sium sisarum Sium sisarum Sium suave Walter Trepocarpus aethusae Nutt. ex DC. GenBank No. Mexico, Oaxaca, Sierra de San Felipe, Molseed 278 (ISU) Hungary; cult. UIUC from seeds obtained from Hungarian Academy of Sciences Bot. Gard., Vácrátót, Downie 476 (ILL) Spain; cult. UIUC from seeds obtained from Real Jardín Botánico, Downie 40 (ILL) Belgium; cult. UIUC from seeds obtained from Jardin Botanique National de Belgique, Downie 247 (ILL) Portugal, Madeira, Levado de Norte, Sunding s.n.; cult. RBGE (no. 19931141) Hungary; cult. Univ. of Oldenburg Bot. Gard, Downie 165 (ILL) Germany; cult. UIUC from seeds obtained from Karl-Marx Univ., Leipzig, Lee 24 (ILL) Belgium; cult. UIUC from seeds obtained from Jardin Botanique National de Belgique, Downie 273 (ILL) Germany; cult. UIUC from seeds obtained from Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Downie 29 (ILL) USA, California, San Mateo Co., Plunkett 1308 (WS) USA, Colorado, Rio Blanco Co., Rough Creek, Vanderhorst 3759 (RM) USA, California, El Dorado Co., Osgood Swamp, Follette s.n. (JEPS) AY360244 AY360245 USA, Illinois, Vermillion Co., Windfall Hill Prairie Nature Preserve, Phillippe et al. 19411 (ILLS) AY360255 USA, Illinois, Shelby Co., NE of Assumption, Shildneck 12868 (ILL) AY246910 USA, California, Sonoma Co., Tin Barn Rd., Raiche 30482 (UC), cult. UC Bot. Gard., Berkeley (no. 83.1080) AY246959 USA, California, Sonoma Co., 5 mi N. of Cazadero, Ornduff et al. s.n. (UC); cult. Univ. of California Bot. Gard. (no. 81. 0521) USA, California, Alameda Co., Berkeley, Univ. of California Bot. Gard., Downie 635 (ILL) USA, California, Sonoma Co., 5 mi N. of Cazadero, Ornduff et al. s.n. (UC); cult. Univ. of California Bot. Gard. (no. 81. 0521) USA, Oklahoma, Rogers Co., Claremore, Jones 3030 (ILL) St Helena, below Cuckhold’s Point on west side, Cronk 32 (E); ITS sequence supplied by J.C. Preston & Q.C.B. Cronk St Helena; material provided by V. Williams s.n. (ILL) St Helena; material provided by V. Williams s.n. (ILL) China, Yunnan, Zhongdian County, Da Xu Shan, RBGE Gyalthang Expedition, FED 109 (E) Denmark, N Sealand, Davis s.n. (E) France, Bas-Rhin, Hultenheim, cult. Conservatoire botanique de la Ville de Mulhouse (no. 9466), Herb. Reduron s.n. Denmark, Sjælland, Bromme Lillesø, Petersen & Seberg GPL31 South Africa, Transvaal, Kaapsche Hoop, Rogers 9101 (G) Hungary; cult. UIUC from seeds obtained from Hungarian Academy of Sciences Bot. Gard., Vácrátót, Downie 97 (ILL) France; cult. Jardin botanique de Caen, Downie 311 (ILL) Canada, Montréal; cult. UIUC from seeds obtained from Jardin botanique de Montréal, Downie 388 (ILL) Spain; cult. UIUC from seeds obtained from Real Jardín Botánico, Downie 53 (ILL) Finland; cult. UIUC from seeds obtained from Univ. Joensuu Bot. Gard., Downie 83 (ILL) Canada, Montréal; cult. UIUC from seeds obtained from Jardin botanique de Montréal, Downie 12 (ILL) USA, Illinois, Alexander Co., Horseshoe Lake Conservation Area, Basinger 10891 (ILLS) AY246962 AY360246 AY360247 AY360248 AY360249 AY360250 U78371 AY360251 AY360252 AY360253 AY360254 AY246961 U78373 AY360256 AY353981, AY353988 AY353982, AY353989 AY353983, AY353990 AF164842, AF164867 AY353984, AY353991 AY360257 AY360258 AY353977 (ITS-1 only) U78370 AY360259 AY360260 AY360261 AY360262 AY360263 AY360264 Hardway, Spalik, Watson, Katz-Downie and Downie 177 178 109 E 1632 2256 61 97 311 388 53 83 12 1817 crocata L. crocata divaricata (R. Br.) Mabb. fistulosa L. peucedanifolia Pollich pimpinelloides L. pimpinelloides Voucher Information South African Journal of Botany 2004, 70: 393–406 Methods Details of the DNA extractions, PCR amplifications and purifications, and DNA sequencing are the same as provided elsewhere (Downie and Katz-Downie 1996, Downie et al. 1998, Hardway 2001). For most accessions, total genomic DNA was obtained from about 20mg of dried, leaf tissue using the Dneasy Plant Mini Kit (Qiagen Inc. Valencia, California). For the remaining accessions, the modified CTAB protocol described by Doyle and Doyle (1987) was used. The purified DNAs were PCR-amplified using primers ‘ITS4’ and ‘ITS5’ (White et al. 1990). Twenty-two complete ITS sequences were obtained through manual sequencing, using the dideoxy chain termination method using Sequenase (version 2.0, United States Biochemical Corporation, Cleveland, Ohio), with a-35S-dATP as the labeling agent. Modifications to the sequencing protocol included denaturation of the DNA by boiling the DNA/primer/ acetamide mix for 4min, followed by snap-chilling the annealing mixture for 3min in an ice water bath. Both PCR primers, and primers ‘ITS2’ and ‘ITS3’ (described in White et al. 1990, including modifications by Downie and KatzDownie 1996), were used in manual sequencing reactions. Cycle sequencing reactions, using primers ‘ITS4’ and ‘ITS5’, were performed on all remaining purified PCR products using AmpliTaq DNA polymerase and fluorescent dyelabeled terminators (ABI Prism BigDye terminator vers. 3.0 Ready Reaction Cycle Sequencing Kit — Applied Biosystems, Foster City, California). Sequencing products were resolved by electrophoresis using Applied Biosystem’s 377A automated DNA sequencer. All ITS sequences have been deposited in GenBank, as either separate ITS-1 and ITS-2 sequences or contiguous ITS-1, 5.8S, ITS-2 data (Table 2). For twelve accessions, sequences from 5.8S rRNA were unavailable, owing to the sequencing methods used to obtain these data. For three of these twelve accessions (Berula erecta nos. 82 and 116, and Sium repandum no. 61), sequence data were also unavailable for ITS-2, despite our repeated but unsuccessful efforts to PCR-amplify this region. Uncorrected pairwise nucleotide differences were determined using PAUP* version 4.0 (Swofford 1998), as they are commonly provided in other angiosperm studies (e.g. Baldwin et al. 1995). Data analysis The DNA sequences were aligned using CLUSTAL X (Jeanmougin et al. 1998). However, given the many small length differences observed among the sequences (particularly for those eight genera comprising the ‘North American (NA) Endemics’ clade, discussed below), a variety of costs for gap opening and gap extension was utilised. As a result of these different alignment parameters, the relative positions of several genera within the ‘NA Endemics’ clade changed, as did the position of this clade relative to the genera Cicuta, Oenanthe, and Oxypolis, when analysed using maximum parsimony. Such changes in tree topologies reflecting different cost matrices have been reported previously for Oenantheae (Petersen et al. 2002). We settled on using the default parameters of CLUSTAL X (specifically, 397 gap opening penalty = 15, and gap extension = 6.66), and reiterate that one of the major goals of this paper is to identify those genera comprising tribe Oenantheae, rather than elucidate all intergeneric relationships (particularly among the taxonomically problematic North American members of the group). The latter will be achieved in subsequent studies, by increasing the sampling of species and incorporating data from the more conservatively evolving chloroplast genome. Moreover, the relationships among Berula, Afrocarum, and the African Sium species, the second major goal of this paper, did not change upon consideration of different gap costs. Relative rate tests, using the method of Robinson et al. (1998), were implemented using the program RRTree version 1.1 (Robinson-Rechavi and Huchon 2000) to detect rate asymmetries of the ITS regions among taxa in tribe Oenantheae. The proportions of site differences were estimated using the two-parameter distance of Kimura (1980). The resulting data matrix was first analysed using maximum parsimony (MP), with gap states treated as missing data. Characters were treated as unordered and all character transformations were weighted equally. Heuristic MP searches were replicated 1 000 times with random stepwise addition of taxa, Tree-Bisection-Reconnection (TBR) branch swapping, and saving Multiple Trees (MulTrees). Bootstrap values were calculated from 1 000 replicate analyses using TBR branch swapping and simple stepwise addition of taxa. The number of additional steps required to force particular taxa into a monophyletic group was examined using the constraint option of PAUP*. The ITS data were analysed as separate ITS-1, 5.8S, and ITS-2 regions, and combined. However, not all data sets were equivalent in their number of terminal taxa, as 5.8S and ITS-2 sequences were unavailable for twelve and three accessions, respectively. To examine the extent of conflict among the ITS-1 and ITS-2 data sets, the incongruence length difference test of Farris et al. (1995) was implemented using PAUP*’s partition-homogeneity test. The test was performed with 100 replicates, using the heuristic search option with simple addition of taxa, and TBR branch swapping. The complete data matrix was then analysed using maximum likelihood, after using the program Modeltest vers. 3.06 (Posada and Crandall 1998) to select an appropriate model of DNA substitution and to estimate its parameters. A heuristic search using random addition sequence and TBR branch swapping was implemented using PAUP*. One thousand bootstrap replicate analyses were conducted using neighbour-joining searches with ML distance, using the ML parameters inferred by Modeltest. All trees were rooted with Perideridia. The results of previous systematic investigations of Apiaceae subfamily Apioideae based on a variety of molecular evidence reveal that the North American genus Perideridia is sister taxon to all other members of the tribe (Plunkett et al. 1996, Downie et al. 1998, 2000a, 2000b). In many studies, the Komarovia clade and tribe Pleurospermeae occur basal to tribe Oenantheae (Downie et al. 2001), rooting the trees with either Komarovia or Pleurospermum maintained Perideridia as sister taxon to all other Oenantheae genera and did little to affect ingroup tree topology (Hardway 2001). 398 Hardway, Spalik, Watson, Katz-Downie and Downie Circumscription of Oenantheae. Phylogenetic analysis of ITS sequences from 100 accessions, representing the 13 core genera of Oenantheae and 19 additional genera examined for possible inclusion in the tribe, resulted in the expansion of tribe Oenantheae by three genera (Afrocarum, Daucosma, and Trepocarpus). Representatives of the 16 remaining genera (Table 1) all fall outside of the tribe; the phylogenetic affinities of each will be discussed in a subsequent paper (K. Spalik and S. Downie, unpubl. data). Bifora americana, the only North American member of the genus, is confirmed as belonging to tribe Oenantheae; its congeners, B. radians M. Bieb. and B. testiculata (L.) Spreng. ex Schult., are placed in the apioid superclade (Downie et al. 2001). Similarly, the genus Cryptotaenia is polyphyletic, with C. africana Drude, C. calycina C.C. Towns., and C. elegans Webb ex Bolle placed outside of tribe Oenantheae, away from C. canadensis (L.) DC. and C. japonica Hassk., which are maintained in the tribe (K. Spalik and S. Downie, unpubl. data). The nomenclatural type of Cryptotaenia (C. canadensis) is included in Oenantheae, whereas the type of Bifora (B. testiculata) is not. In summary, 17 genera are recognised herein as constituting tribe Oenantheae. Sequence analysis Alignment of 64 ITS sequences, representing all 17 genera of tribe Oenantheae, resulted in a matrix of 633 positions, with three positions near the ITS-2–26S rRNA boundary excluded because of alignment ambiguity. Characteristics of these aligned data, as separate or combined ITS-1, 5.8S, and ITS-2 regions, are presented in Table 3. Fifty-eight unambiguous gaps, all but one ranging between one and three bp in size, were introduced to facilitate alignment. The remaining and largest gap, of 19 bp in size, characterised all accessions of Helosciadium. Of these 58 gaps, single bp deletions (relative to the Perideridia sequences) were most numerous (27), followed by single bp insertions (16) and two bp insertions (8; Figure 1). Half of these 58 gaps were restricted to sequences from the eight species comprising the ‘NA Endemics’ clade (discussed below). A total of 31 gaps was parsimony informative; these were distributed almost equally between both spacer regions. Treating gaps as missing data, uncorrected pairwise sequence divergence values across the entire region ranged from identity (for several conspecific taxa) to 26.9% of nucleotides (between Lilaeopsis occidentalis and Helosciadium inundatum). The vast majority of pairwise comparisons ranged between 6% and 14%, whereas the highest divergence values were obtained among pairwise comparisons of sequences from the eight ‘NA Endemics.’ For the latter, these values ranged between 5.7% and 20.5% (and averaged 17%). Phylogenetic analysis MP analysis of combined ITS-1, 5.8S, and ITS-2 sequence data for 64 accessions of Apiaceae tribe Oenantheae resulted in 256 minimal length trees, each of 999 steps (Consistency Indices (CI’s) = 0.5315 and 0.4846, with and without uninformative characters, respectively; Retention 40 N um ber ofgaps Results 30 Insertion D eletion 20 10 1 2 3 19 Size ofgaps (bp) Figure 1: The number of gaps and their sizes inferred in the alignment of 64 ITS sequences of Apiaceae tribe Oenantheae. The number of insertions relative to deletions is indicated Table 3: Comparisons among the data sets and most parsimonious (MP) trees presented in this study. The number of terminal taxa varied, for 5.8S and ITS-2 data were unavailable for 12 and 3 accessions, respectively Data Set Characteristics and Cladogram Measures No. of terminals Length variation (bp) No. of aligned positions No. of aligned positions excluded No. of aligned positions constant No. of aligned positions autapomorphic No. of aligned positions parsimony informative No. of unambiguous alignment gaps No. of alignment gaps parsimony informative Pairwise sequence divergence (range in %) No. of MP trees Length of MP trees Consistency index Consistency index (excluding uninformative chars.) Retention index ITS–1 64 208–213 224 0 84 32 108 27 15 0–26.4 1 376 432 0.5231 0.4824 0.7832 5.8S 52 161–164 164 0 146 8 10 4 3 0–4.4 >5 000 27 0.7037 0.5789 0.8889 ITS–2 61 207–232 245 3 66 40 136 27 13 0–32.4 348 510 0.5608 0.5141 0.7887 Combined 64 580–606 633 3 296 80 254 58 31 0–26.9 256 999 0.5315 0.4846 0.7752 South African Journal of Botany 2004, 70: 393–406 399 74 68 77 95 62 89 99 100 71 100 98 99 100 99 82 100 69 100 99 99 100 100 100 100 42 96 81 60 88 90 32 94 48 75 99 63 32 92 100 98 71 100 71 Berula erecta 82 Berula erecta 116 Berula erecta 799 Sium bracteatum Sium bracteatum 177 Sium burchellii 178 Afrocarum imbricatum 132 Afrocarum imbricatum 1444 Sium repandum 61 Berula erecta 150 Berula erecta 2257 Berula erecta 2253 Berula erecta 251 Helosciadium nodiflorum 919 Helosciadium nodiflorum 15108 Helosciadium nodiflorum 1871 Helosciadium bermejoi 62449 Helosciadium repens 1870 Helosciadium inundatum 64358 Helosciadium crassipes 170 Sium sisarum 97 Sium sisarum 311 Sium sisarum 388 Sium sisarum 83 Sium sisarum 53 Sium frigidum 109 Sium latifolium E Sium latifolium 1632 Sium latifolium 2256 Sium suave 12 Cryptotaenia japonica 402 Cryptotaenia japonica 574 Cryptotaenia canadensis 817 Oenanthe pimpinelloides 273 Oenanthe pimpinelloides 29 Oenanthe banatica 476 Oenanthe fistulosa 165 Oenanthe peucedanifolia 1282 Oenanthe divaricata 1300 Oenanthe crocata 40 Oenanthe crocata 247 Oenanthe sarmentosa 521 Limnosciadium pinnatum 2000 Daucosma laciniata 2397 Ptilimnium capillaceum 2165 Cynosciadium digitatum 1063 Lilaeopsis occidentalis 1999 Trepocarpus aethusae 1817 Bifora americana 1467 Neogoezia minor 2138 Cicuta virosa 131 Cicuta virosa 75 Cicuta virosa 426 Cicuta maculata var. angustifolia 1113 Cicuta bulbifera 1923 Cicuta bulbifera 1935 Oxypolis occidentalis 1142 Oxypolis rigidior 1927 Oxypolis fendleri 915 Perideridia kelloggii 855 Perideridia kelloggii 635 Perideridia kelloggii 778 Perideridia americana 1938 Perideridia howellii 780 “Berula” Helosciadium Sium Cryptotaenia Oenanthe “NA Endemics” Cicuta Oxypolis Perideridia Figure 2: Strict consensus of 256 maximally parsimonious 999-step trees derived from equally weighted maximum parsimony analysis of aligned ITS-1, 5.8S, and ITS-2 sequences from 64 accessions of Apiaceae tribe Oenantheae (CI = 0.4846, excluding uninformative characters; RI = 0.7752). Numbers on branches represent bootstrap percentage estimates from 1 000 replicate analyses. Brackets indicate clade descriptors discussed in the text 400 Index (RI) = 0.7752 (Table 3). The strict consensus of these trees is presented in Figure 2. Separate MP analyses of the ITS-1 and ITS-2 data sets resulted in strict consensus trees (not shown) slightly less resolved but highly consistent with the strict consensus tree derived from combined data. The results of the partition-homogeneity test revealed that the two spacer regions do not yield significantly different phylogenetic estimates. Separate analysis of the 5.8S region resulted in a large polytomy, with only the group of eight ‘NA Endemic’ genera resolved as monophyletic. Greatest resolution of relationships was achieved when all molecular data were considered together, a result concordant to that reported from other studies of ITS data (Baldwin et al. 1995). Of the 31 potentially informative alignment gaps, 16 mapped without homoplasy when optimised on all minimal length trees. The largest gap, restricted to all Helosciadium sequences, was a 19bp deletion relative to the outgroup Perideridia. Other synapomorphic indels supported the monophyly of the genera Cicuta, Cryptotaenia pro parte (i.e. C. canadensis and C. japonica), Oxypolis, and Perideridia, and the species groups Sium bracteatum + Sium burchellii, Afrocarum imbricatum + Sium repandum, Sium latifolium + Sium suave, and Trepocarpus aethusae + Bifora americana. On the basis of these results, the genera Helosciadium, Cryptotaenia pro parte (as above), Oenanthe, Cicuta, Oxypolis, and Perideridia constitute well-diagnosed groups, with supporting bootstrap values ranging between 92% and 100% and the possession of uniquely occurring indels. The genera Berula and Sium are not monophyletic. The two St Helena endemics (Sium bracteatum and S. burchellii) ally with the three Berula accessions from Africa (nos. 82, 116, and 799). This clade is sister to Afrocarum imbricatum + Sium repandum, two species also native to Africa, which, in turn, comprise a clade sister to the four accessions of European Berula examined (nos. 150, 251, 2253, and 2257). This entire clade, labeled ‘Berula’, comprising Afrocarum, Berula and the three Sium species endemic to St Helena and continental Africa, is supported strongly, with a bootstrap value of 100%. Sium sisarum (five accessions) + S. frigidum and Sium latifolium (three accessions) + S. suave comprise two distinct clades arising from a fivebranched polytomy, along with the ‘Berula’ clade, Helosciadium, and Cryptotaenia (pro parte). Constraining the seven examined accessions of Berula erecta to monophyly and rerunning the MP analysis resulted in trees three steps longer than those most parsimonious. Constraining the ten non-African Sium accessions to monophyly (i.e. Sium sisarum, S. frigidum, S. latifolium, and S. suave) revealed a subset (224) of the 256 minimal length 999-step trees resulting from unconstrained analysis. Constraining all 14 Sium accessions to monophyly (including the African Sium bracteatum, S. burchellii and S. repandum) resulted in trees 26 steps longer than those most parsimonious. Based on these results, it is very unlikely that the genus Sium, as presently circumscribed to include the three African species, is monophyletic. In contrast, Berula erecta may prove to be monophyletic upon subsequent study and expanded sampling, given the many weakly supported internal branches in this portion of the tree. The last major clade in the MP tree, labeled ‘NA Hardway, Spalik, Watson, Katz-Downie and Downie Endemics’, comprises eight species native to North America. The genera Cynosciadium, Daucosma, Limnosciadium, Ptilimnium, and Trepocarpus are found exclusively in the USA, as is Bifora americana. Neogoezia is endemic to Mexico (Constance 1987). Lilaeopsis occidentalis is almost entirely confined to the Pacific coast of North America, whereas the genus itself is distributed more widely in the temperate regions of North and South America, with a few outlying species in Australasia and elsewhere (Affolter 1985, Petersen and Affolter 1999). While we refer to this group as the ‘NA Endemics’ clade, we acknowledge that there are taxa outside of the clade that are also endemic to North America (such as Oxypolis, Perideridia, and all but one species of Cicuta). We also acknowledge that very few species of Lilaeopsis are actually native to North America. Therefore, we use the descriptor ‘NA Endemics’ for the sake of reference only. Based on the results of the hierarchical likelihood ratio tests, Modeltest selected the TrN+G model of nucleotide substitution (Tamura and Nei 1993) as fitting these ITS data best (base frequencies: 0.2416, A; 0.2258, C; 0.2446, G; 0.2879, T; estimates of substitution rates: A↔C, 1; A↔G, 2.1879; A↔T, 1; C↔G, 1; C↔T, 4.4819; G↔T, 1; proportion of invariable sites = 0; gamma distribution shape parameter = 0.5083). Using these parameters, a single tree was recovered in PAUP*, with a –Ln likelihood score of 5805.7183 (Figure 3). A tree with identical topology (with a –Ln likelihood score of 5792.08846) was recovered using the best-fit model GTR+I+G (Rodríguez et al. 1990; proportion of invariable sites = 0.2017; gamma distribution shape parameter = 0.8653), selected by Modeltest’s Akaike information criterion (Akaike 1974). The results of the ML analyses are similar to those inferred by MP, with the following exceptions: Oenanthe and Cicuta arise as weakly supported sister taxa; the ten non-African Sium accessions (Sium sisarum, S. frigidum, S. latifolium, and S. suave) unite as a weakly supported monophyletic group (with a 66% bootstrap value); and decreased internal support within the ‘Berula’ clade, including the near collapse of the branch uniting African Berula, Sium, and Afrocarum. The latter clade, however, is still supported strongly, with a 100% bootstrap value. The presence of a five-branched polytomy in the MP tree, the many weakly supported or short basal branches in both MP and ML trees, and the rearrangement of certain taxa in the ‘NA Endemics’ clade depending upon the gap penalties invoked in generating the alignment, generally preclude unambiguous hypotheses of intergeneric relationship within tribe Oenantheae. Those relationships that are noteworthy include the union of Afrocarum with Sium repandum, the close affinity between the jellicos (i.e. Sium bracteatum and S. burchellii) of St Helena and African Berula, and the isolation of African Sium from its north temperate congeners, such as Sium latifolium, the nomenclatural type of the genus. A striking feature of the ITS trees is the relatively long branch lengths characterising the members of the ‘NA Endemics’ clade, as seen in Figure 3. Sequence divergence values among the eight members comprising this clade are approximately 6–7 times higher (averaging 17%) relative to those within Cicuta (averaging 2.4%) or Oenanthe (averag- South African Journal of Botany 2004, 70: 393–406 401 Sium sisarum 97 Sium sisarum 311 0.01 substitutions/site Sium sisarum 388 80 Sium sisarum 83 Sium sisarum 53 Sium 66 Sium frigidum 109 100 Sium latifolium E Sium latifolium 1632 97 Sium latifolium 2256 Sium suave 12 Helosciadium nodiflorum 919 Helosciadium nodiflorum 15108 Helosciadium nodiflorum 1871 Helosciadium Helosciadium bermejoi 62449 100 Helosciadium repens 1870 Helosciadium inundatum 64358 Helosciadium crassipes 170 54 69 Berula erecta 82 Berula erecta 116 60 Berula erecta 799 89 Sium bracteatum Sium bracteatum 177 Sium burchellii 178 “Berula” Afrocarum imbricatum 132 94 61 Afrocarum imbricatum 1444 100 Sium repandum 61 Berula erecta 2257 Berula erecta 2253 96 Berula erecta 150 Berula erecta 251 97 Cryptotaenia japonica 402 97 Cryptotaenia Cryptotaenia japonica 574 Cryptotaenia canadensis 817 Oenanthe pimpinelloides 273 100 Oenanthe pimpinelloides 29 Oenanthe banatica 476 Oenanthe fistulosa 165 Oenanthe Oenanthe peucedanifolia 1282 100 Oenanthe sarmentosa 521 100 Oenanthe divaricata 1300 Oenanthe crocata 40 Oenanthe crocata 247 virosa 131 100 Cicuta Cicuta virosa 75 Cicuta virosa 426 98 Cicuta Cicuta maculata var. angustifolia 1113 98 Cicuta bulbifera 1923 Cicuta bulbifera 1935 92 Limnosciadium pinnatum 2000 67 Daucosma laciniata 2397 84 Ptilimnium capillaceum 2165 96 “NA Cynosciadium digitatum 1063 Endemics” Lilaeopsis occidentalis 1999 89 51 96 Trepocarpus aethusae 1817 Bifora americana 1467 Neogoezia minor 2138 64 Oxypolis occidentalis 1142 99 Oxypolis rigidior 1927 Oxypolis Oxypolis fendleri 915 Perideridia kelloggii 855 100 Perideridia kelloggii 635 79 Perideridia Perideridia kelloggii 778 Perideridia americana 1938 Perideridia howellii 780 94 Figure 3: The single tree derived from maximum likelihood analysis of aligned ITS-1, 5.8S, and ITS-2 sequences from 64 accessions of Apiaceae tribe Oenantheae under a TrN+G model of nucleotide substitution (–Ln likelihood = 5805.7183). Numbers on branches represent bootstrap estimates for 1 000 replicate neighbor-joining analyses using a maximum likelihood model of nucleotide substitution; bootstrap percentage estimates <50% are not indicated. Brackets indicate clade descriptors discussed in the text 402 ing 2.8%), their putative sister taxa. Moreover, half of the 58 gaps inferred in the multiple alignment of all 64 ITS sequences were restricted to members of the ‘NA Endemics’ clade, as was the single small region of ambiguous alignment near the ITS-2–26S rRNA boundary excluded from the analysis. To detect rate asymmetry, 28 relative rate tests were conducted. Twelve sequences were assigned to nine defined lineages (representing one sequence from each of the nine major clades outlined in Figures 2–3, with the exception of the ‘NA Endemics’ clade, where four sequences were assigned). Perideridia kelloggii (no. 635) was used as the reference taxon (outgroup). Significant differences (P = 0.001) suggest that Limnosciadium, Ptilimnium, Lilaeopsis, and Neogoezia, the four examined sequences from the ‘NA Endemics’ clade, are each evolving much faster when compared to any sequence from outside of this clade. Rate differences of most other pairs of species were not statistically significant (at the 5% level). The molecular clock hypothesis for Oenantheae ITS sequences is therefore rejected. Discussion The circumscription and distribution of Apiaceae tribe Oenantheae Table 4 lists the 17 genera recognised here as comprising tribe Oenantheae and their distributions. Four genera are not monophyletic. Bifora and Cryptotaenia have members that fall outside of the tribe; Berula and Sium each comprise two or more lineages within Oenantheae. The separation of Bifora americana from its Eurasian congeners, B. radians and B. testiculata, the latter the nomenclatural type of the genus, involves a change in nomenclature. The name Hardway, Spalik, Watson, Katz-Downie and Downie Atrema americana DC. already exists for these North American plants, but further study of North American Oenantheae is in order before such a change is implemented. The type of Cryptotaenia, C. canadensis, is maintained within the tribe, as is C. japonica; the latter, however, depending upon the treatment, may be recognised as a variety or subspecies of the former. Two African species of Cryptotaenia (C. africana and C. calycina) and the Macaronesian C. elegans, coinciding with Wolff’s (1927) section Afrosciadium, are excluded from the tribe. Information on their phylogenetic placements is forthcoming (K Spalik and S Downie, unpubl. data). The western North American monotypic genus Shoshonea Evert & Constance, erroneously placed in the Oenanthe clade on the basis of matK sequence comparisons (Plunkett et al. 1996), belongs in the ‘Angelica’ clade of the apioid superclade (Downie et al. 1998, 2001, Plunkett and Downie 1999). These plants are caespitose-pulvinate, scaberulous, and possess a woody taproot, and are morphologically similar to several other genera of the region (Downie et al. 2002). They are also restricted to exposed calcareous outcroppings at high elevations (Evert and Constance 1982). Any of these features would make this genus an anomaly, if it was maintained in tribe Oenantheae. The matK study of Plunkett et al. (1996) also placed Cicuta (specifically, C. douglasii (DC.) J.M. Coult. & Rose) in the Angelica clade, alongside three genera of North American distribution having affinities with Shoshonea (Downie et al. 2002). The genus Cicuta is unequivocally monophyletic (C. Lee and S. Downie, unpubl. data), and its position outside of tribe Oenantheae should be regarded as spurious. Nine genera are native to North America (six exclusively to the USA), of which five are monotypic or bitypic. Three Table 4: The composition and distribution of Apiaceae tribe Oenantheae Dumort. Species numbers are after Pimenov and Leonov (1993), except for Berula (Burtt 1991), Cicuta (Mulligan 1980), Cynosciadium (Mathias and Constance 1944-1945), Helosciadium (Wolff 1927; Z Popper and M Watson, unpubl. data), and Lilaeopsis (Affolter 1985, Petersen and Affolter 1999). Asterisks denote those genera that are not monophyletic as a result of this study Genus Afrocarum Rauschert Berula W.D.J. Koch* Bifora Hoffm.* Cicuta L. Cryptotaenia DC.* Cynosciadium DC. Daucosma Engelm. & A. Gray ex A. Gray Helosciadium W.D.J. Koch Lilaeopsis Greene Limnosciadium Mathias & Constance Neogoezia Hemsl. Oenanthe L. Oxypolis Raf. Perideridia Rchb. Ptilimnium Raf. Sium L.* Trepocarpus Nutt. ex DC. a No. of Species 1 1 1a 4 2b 1 1 5 14 2 5 40 7 13 5 14 1 Distribution Africa Widespread North America (USA) 3 NA; 1 Circumboreal Widespread North America (USA) North America (USA) Europe New World, Australasia North America (USA) Mexico Widespread North America North America North America (USA) Widespread North America (USA) Bifora americana (=Atrema americana DC.). Bifora radians and B. testiculata are excluded from tribe Oenantheae Cryptotaenia canadensis and C. japonica. Cryptotaenia africana, C. calycina, and C. elegans are excluded from tribe Oenantheae. The phylogenetic placements of C. flahaultii Koso-Pol., C. polygama C.C. Towns., and C. thomasii (Ten.) DC. have yet to be determined b South African Journal of Botany 2004, 70: 393–406 403 species of Cicuta are also confined to North America, whereas C. virosa is circumboreal (Mulligan 1980). Afrocarum is endemic to tropical Africa, and Helosciadium is European in distribution. Lilaeopsis occurs in the temperate zones of North and South America and Australasia (Affolter 1985), as well as in Mauritius in the southwest Indian Ocean (Petersen and Affolter 1999). The remaining four genera are widely distributed, occurring in Europe, Asia, Africa, North America and, depending upon which genus, also in Central America, Australia and Australasia (Pimenov and Leonov 1993). Moreover, at least four new name combinations will be necessary. Another approach, equally unwieldy, is to recognise Afrocarum, Sium repandum, the St Helena endemics, and African Berula as separate genera. With the exception of the latter, each can be circumscribed unequivocally because of their distinctive morphology, but this leads to the creation of several monotypic genera, of which there are already far too many in the family (Spalik et al. 2001). However, before any such nomenclatural changes are implemented, further sampling and study are required, especially of Berula erecta. The ‘Berula’ clade The ‘NA Endemics’ clade A well-supported clade in all trees (the ‘Berula’ clade) contains Berula erecta, Afrocarum imbricatum, Sium bracteatum, S. burchellii, and S. repandum. Burtt (1991) recognised two subspecies within Berula erecta, and established subsp. thunbergii (DC.) B.L. Burtt ‘with some reluctance’. These two subspecies are separated by the severity of cutting of the leaflets of the cauline leaves, with subsp. thunbergii having a more regular and less deeply dentate cutting than that of the typical subspecies. Moreover, subsp. erecta, although distributed widely in temperate Eurasia, North America, and elsewhere, does not occur in southern Africa (Burtt 1991). Geographic distribution aside, we could not satisfactorily distinguish between these subspecies, because one of our accessions from Africa had a jagged leaflet morphology just like European Berula. Nevertheless, it is intriguing that the African accessions of Berula erecta comprise a distinct clade, separate from their European counterparts. Therefore, while our results show that subsp. thunbergii may be a distinct taxon, the diagnostic characters used to distinguish it from the typical subspecies appear to be incorrect. The jellicos of St Helena, Sium bracteatum and S. burchellii, unite as a monophyletic group sister to the three African Berula accessions. In both MP and ML trees, this entire group is sister to the clade of Afrocarum imbricatum and Sium repandum, suggesting that the St Helena endemics originated from Africa and shared an immediate common ancestor with African Berula. The four examined accessions of B. erecta from Europe comprise a strongly supported monophyletic group sister to the clade of all other aforementioned taxa. This alliance among Berula, Sium, and Afrocarum is not surprising. Berula and Sium are morphologically very similar and, in many early systems of classification, were treated as congeneric. The genus Afrocarum resembles Sium in several attributes (Cannon 1978), although it is generally, but erroneously, affiliated with Carum, under which it was first described (Cannon 1978, Townsend 1989). It is also intriguing that the jellicos of St Helena and Sium repandum are not immediately allied to their north-temperate congeners, such as Sium latifolium, the nomenclatural type of the genus. The phylogenetic results presented here indicate that nomenclatural changes are in order, especially with regard to the monotypic genera Afrocarum and Berula vis-à-vis Sium. One approach is to redefine Berula to include Afrocarum, Sium repandum, and the two St Helena Sium species, but this leads to a complex genus that, pending further study, cannot readily be circumscribed morphologically. Based on ITS sequence data, all members of the ‘NA Endemics’ clade show a high level of sequence divergence, and relative rate tests suggest that this lineage is evolving much faster than any other within the tribe. This rapid divergence parallels their great morphological diversity. For example, many members of this clade (including the genus Lilaeopsis) exhibit a severe reduction of leaf morphology (Affolter 1985). Others, such as Bifora americana and Trepocarpus, have pinnately decompound leaves with filiform divisions. Their distinctive morphology has confused phylogenetic placement; as examples, Neogoezia and Lilaeopsis, with their simple umbels, have been treated in both subfamilies Apioideae and Hydrocotyloideae (reviewed in Constance 1987, and Peterson et al. 2002). Further study of the ‘NA Endemics’ clade is currently underway. Taxonomic history The taxonomic history of tribe Oenantheae Dumort. (as emended by Downie et al. 2000b and expanded upon here) is extraordinarily complex, especially because of the use of many longstanding names in earlier works that are now considered as synonyms. Sprengel (1820), proposing the first formal (i.e. tribal) subdivisions of subfamily Apioideae, placed Cicuta in tribe Smyrnieae Spreng., Sium in tribe Ammieae Spreng., and Oenanthe in tribe Pimpinelleae Spreng. Koch (1824), emphasising features of the seed face (endosperm) and mericarp ribs, moved Oenanthe into tribe Seseleae W.D.J. Koch and Cicuta into tribe Ammieae to join Sium, Helosciadium, and Drepanophyllum Hoffm. (the latter two genera being segregates of Sium). Dumortier (1827) refined Koch’s treatment by placing Cicuta, Helodium Dumort. (= Helosciadium), and Sium in tribe Pimpinelleae subtribe Cicutinae Dumort. on the basis of the presence of flat endosperm and calyx teeth. In the same publication, Dumortier described the new tribe Oenantheae for the genera Aethusa L., Coriandrum L., and Oenanthe, defined by the presence of radiately ribbed fruits. This rather heterogeneous assemblage of genera was not followed by later authors, nor is it supported by molecular studies. De Candolle (1830), following Koch’s emphasis on the shape of the endosperm, as well as the distribution of vittae and other fruit anatomical characters, treated Cicuta, Helosciadium, and Sium (incl. Berula) in tribe Ammieae. He also included in this tribe, along with 18 other genera, his newly described Cryptotaenia DC. and Discopleura DC., the latter now treated in Ptilimnium. Oenanthe (including 404 Dasyloma DC.) and Cynosciadium were placed in Seseleae, with this tribe distinguished from Ammieae on the basis of the degree and direction of fruit compression. De Candolle treated his new genera Archemora DC. (= Oxypolis) and Tiedemannia DC. (= Oxypolis) in tribe Peucedaneae, Eulophys Nutt. ex DC. (= Perideridia) in tribe Smyrnieae, Trepocarpus Nutt. ex DC. in tribe Cumineae W.D.J. Koch, and Crantzia Nutt. (= Lilaeopsis), with its greatly reduced vegetative morphology and simple umbels, in tribe Hydrocotyleae Spreng. As such, the genera we recognise here as comprising tribe Oenantheae were scattered among six tribes! The system of Bentham (1867) departed from all previous treatments, as he emphasised inflorescence form and the presence or absence of fruit vittae and secondary ribs. Once more, Cicuta, Cryptotaenia, and Sium (incl. Berula) were placed together in Ammieae subtribe Ammiinae Dumort.; however, Oenanthe, Cynosciadium, and Discopleura (= Ptilimnium) were treated in Seseleae subtribe Oenanthinae Benth. Also included in the latter was Crantzia (= Lilaeopsis), and eight other genera of distant affinity. Eulophus (= Perideridia) was maintained in Smyrnieae, but Trepocarpus was placed in tribe Caucalideae Spreng., along with Daucus and, surprisingly, Bifora. In the system of Boissier (1872), Oenanthe was placed in tribe Seseleae, away from Helosciadium, Sium, and Berula of tribe Ammieae. Drude (1898) maintained two major groups of genera, with Cicuta, Cryptotaenia, Trepocarpus, and Sium (incl. Berula) in Ammieae subtribe Carinae Drude, and Oenanthe, Cynosciadium, Crantzia (= Lilaeopsis), and Discopleura (= Ptilimnium) in Ammieae subtribe Seselinae Benth. Eulophus (= Perideridia) was moved to subtribe Carinae, and Helosciadium was included within an expanded Apium. Tiedemannia (= Oxypolis) was maintained in Peucedaneae. The exclusively Mexican genus Neogoezia was placed in Smyrnieae. Pimenov and Leonov (1993), basing their system on that of Drude, placed all but two of these genera into their large, heterogeneous tribe Apieae. Neogoezia and Oxypolis were maintained in Smyrnieae and Peucedaneae, respectively. The detailed fruit anatomical studies of Koso-Poljansky (1916, 1917) only included some of the genera of our Oenantheae, but even so they were widely scattered among several tribes (for example, Helosciadium in Aethuseae Koso-Pol.; Sium in Crithmeae Koso-Pol.; Oxypolis in Peucedaneae; and Cicuta, Oenanthe, Trepocarpus, Cyssopetalum Turcz. (= Oenanthe), and Ptilimnium, along with 12 other genera now considered not very closely related, in Oenantheae Dumort.). Similarly, the novel groupings proposed by Cerceau-Larrival (1962), from her study of pollen and seedling morphology, and later adopted by Guyot (1966, 1971) in his survey of stomatal types, have done little to clarify the relationships among present-day Oenantheae. Cerceau-Larrival placed Berula, Apium (incl. Helosciadium), and Sium in her tribe Heteromorpheae, Cryptotaenia in her tribe Cryptotaenieae, and Oenanthe in a monotypic Oenantheae. None of her names, however, were validly published. In summary, no prior taxonomic treatment has unambiguously grouped together those genera defined herein as belonging to tribe Oenantheae. In recognising the Hardway, Spalik, Watson, Katz-Downie and Downie tribe, Downie et al. (2000b) used Dumortier’s (1827) name, but its circumscription is radically different from that proposed previously. Final considerations In this study, we considered representatives of 19 genera (beyond those recognised in Oenantheae at the onset of this study) for possible inclusion in the tribe. Of these, three (Afrocarum, Daucosma and Trepocarpus) represent the most recent additions to tribe Oenantheae. Two genera (Bifora and Cryptotaenia) were confirmed as polyphyletic, with some of their members having affinities outside of the tribe. The North American Bifora americana may be recognised as Atrema americana, pending further investigation. Molecular systematic studies have revealed that many genera of Apioideae are polyphyletic (Downie et al. 2001, Spalik et al. 2001); thus it is not unrealistic to assume that as material from additional species becomes available for those 16 genera examined for inclusion in the tribe but excluded on the basis of phylogenetic analysis of ITS data (such as, Pimpinella and its segregates), some may find affinity within tribe Oenantheae. Additional genera whose fruit and (or) vegetative morphologies suggest that they should be examined for possible inclusion in tribe Oenantheae include Apodicarpum Makino, Asciadium Griseb., Kundmannia Scop., Ottoa Kunth, and Rutheopsis A. Hansen & G. Kunkel. Apium sensu lato also merits special consideration. In the most recent worldwide treatment of Apium, Wolff (1927) recognised five sections, with one of these comprising those species recognised in the oenanthoid genus Helosciadium. Section Apium is taxonomically complex, and given the vegetative similarity of some of its members with those of tribe Oenantheae, it also deserves further attention. Lastly, while the ITS region provides phylogenetic resolution in tribe Oenantheae, additional sources of phylogenetic information from molecules and morphology, and denser taxonomic sampling are needed to gain a comprehensive and detailed phylogenetic understanding of Apiaceae tribe Oenantheae. Acknowledgements — The authors thank Jean-Pierre Reduron, Vincent Williams, and Gitte Petersen, and the many botanic gardens and herbaria cited in the text, for generously providing us with plant material. We also thank Byoung Yoon Lee, Jennifer Anderson, and Feng-Jie Sun for laboratory assistance, Jill Preston and Quentin Cronk for ITS data, and Carolina Calviño and Pieter Winter for comments. Lastly, we thank Patricia Tilney and Ben-Erik van Wyk for organising this symposium. 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