Bot. Jahrb. Syst. 125 1 19–51 Stuttgart, 22. Dezember 2003 A systematic analysis of Heliotropiaceae (Boraginales) based on trnL and ITS1 sequence data By Hartmut H. Hilger and Nadja Diane With 10 figures and 2 tables Abstract HILGER, H. H. & DIANE, N.: A systematic analysis of Heliotropiaceae (Boraginales) based on trnL and ITS1 sequence data. — Bot. Jahrb. Syst. 125: 19–51. 2003. — ISSN 0006-8152. The infrafamilial relationships of Heliotropiaceae (Boraginaceae subfam. Heliotropioideae according to traditional systems) are reevaluated using molecular data of nuclear ITS1 (86 species) and plastidal trnLUAA intron (66 species) sequences. The results obtained from our investigations show that traditional generic limits warrant adjustment. Heliotropiaceae fall into two large clades. The first clade includes, in basal position, the genus Ixorhea. The genus Myriopus (formerly Tournefortia sect. Cyphocyema) is sister to Euploca (formerly Hilgeria, Schleidenia, Heliotropium sect. Orthostachys). The remaining sections of Heliotropium, Tournefortia sect. Tournefortia and the three small genera Argusia, Ceballosia, and Nogalia, segregated from Heliotropium , constitute the second large clade. Argusia, Ceballosia, and Nogalia cluster within clades of Heliotropium and therefore are reincluded in this genus. Within Heliotropium, the species of former Tournefortia sect. Tournefortia represent a lineage of tropical New World Heliotropium species, growing in humid environments, whereas all other Heliotropium species are found in semi-arid habitats. Before new combinations in the genus Heliotropium are made for “Tournefortia”, the exact relationship within New World Heliotropium needs to be resolved, and a revision of “Tournefortia” is inevitable. We advocate maintaining the genus Tournefortia, which is easily defined, and we conclude that under this definition the genus Heliotropium is paraphyletic. Five genera are thus accepted; 22 new combinations within Heliotropiaceae are presented. Keywords: Boraginaceae, Boraginales, Heliotropiaceae, ITS1, molecular systematics, trnL intron. DOI: 10.1127/0006-8152/ 2003/0125-0019 0006-8152/ 03/0125-0019 $ 08.25 © 2003 E. Schweizerbart’sche Verlagsbuchhandlung, D-70176 Stuttgart 20 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Introduction This study expands upon previous investigations (DIANE et al. 2002a) in evaluating intrafamilial relationships of Heliotropiaceae, based upon molecular and morphological data. In Heliotropiaceae generic limits have fluctuated dramatically. Problems in classifying Heliotropiaceae originated, at least in part, from LINNÉ (1753), with generic definitions being largely to exclusively based on fruit morphology. The two large core-genera have always been maintained, namely Heliotropium and Tournefortia, but various segregates have been discussed controversially (CANDOLLE 1845, GÜRKE 1893, JOHNSTON 1935a). FÖRTHER (1998) recognized a total of ca. 450 species in Heliotropiaceae (as Heliotropioideae). Besides Heliotropium L. and Tournefortia L., he accepted Argusia Böhm., Schleidenia Endl. and the monotypic genera Ceballosia (L.f.) Kunkel ex Förther, Ixorhea Fenzl, as well as Nogalia Verdc. Furthermore, he erected the new genus Hilgeria Förther, comprising three aberrant species from the West Indies, formerly included in Heliotropium. The small segregate genera differ mainly in fruit morphology (mostly dry vs. drupaceous) and habit from the two large genera, which otherwise comprise an enormous range of morphological characters. DIANE et al. (2002a) described the relationship among Heliotropium, Tournefortia, Schleidenia, Ixorhea, and Ceballosia, and pointed out that Tournefortia and Heliotropium are not monophyletic. This study includes data from additional taxa within the genus Hilgeria, as well as Argusia (two Asian species) and the monotypic African genus Nogalia. Furthermore, we include further representatives of Heliotropium and Tournefortia, covering the entire geographical and ecological range. For sequence analysis we use the nuclear internal transcribed spacer region (ITS1) and the plastidal trnLUAA intron. Materials and methods Sampling We sequenced the plastidal trnLUAA intron of 66 species of Heliotropiaceae. 49 new nuclear ITS1 sequences of Heliotropiaceae were added to 37 ITS1 sequences already published by DIANE et al. (2002a). Samples used for molecular analyses were obtained from either silica dried material, fresh material, or herbarium material. We used one species each of Hydrophyllaceae and Ehretiaceae for outgroup comparison. Sources of plant material, used in this analysis are shown in Table 1 together with GenBank accession numbers. DNA extraction, amplification, and sequencing DNA was isolated using a modified CTAB (cetyltrimethylammonium bromide) extraction protocol from DOYLE & DOYLE (1990), amplificated and sequenced as described in DIANE et al. (2002a). The PCR primers for the chloroplast DNA (cpDNA) trnL intron correspond to TABERLET et al. (1991), and the ITS1 primers were those used by BALDWIN (1992). 21 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Tab. 1. The species investigated in this survey, including the location of voucher specimens. DNA-numbers correspond to the BSB. (BSB) = Institut für Biologie — Systematische Botanik und Pflanzengeographie, Freie Universität Berlin, Germany; (CANB) = Australian National Herbarium, Canberra, Australia; (FR) = Herbarium Senckenbergianum, Frankfurt, Germany; (KAS) = Herbarium University Kassel, Germany; (M) = Botanische Staatssammlung München, Germany; (MO) = Missouri Botanical Garden Herbarium, USA. Species Voucher Source BSB GenBank accession accession number number trnL GenBank accession number ITS1 OUTGROUP Nama demissum A.Gray Hofmann 10/98 (BSB) USA: California 281 AY376168 AF402590 Ehretia acuminata R.Br. cult. Botanical Garden of Adelaide (BSB) Australia 492 AY376167 AF385798 Argusia sibirica (L.) Dandy as Tournefortia. sibirica L. Skvortsov s.n. 02.06.1962 (M) Russia 893 AY376169 AY377789 Argusia sogdiana (Bunge) Czerep. as Heliotropium argusioides Kar. & Kir. Belianina & Sofeikiva 11543 (M) Russia 773, 40 AY376170 AY377790 Ceballosia fruticosa (L.f.) Kunkel ex Förther (as Tournefortia messerschmidia Sweet) cult. Royal Botanic Gardens, Kew, Great Britain Spain: Canary Islands 303 AY376171 AY377791 Heliotropium adenogynum I.M.Johnst. Cano 10058 (M) Peru 726 AY376172 AY377792 Heliotropium aegyptiacum Lehm. Schultka 1995/5 (BSB) Kenya 16 AY376173 AF396918 Heliotropium amplexicaule Vahl Hilger Arg_95/70 (BSB) Argentina 59 AY376174 — Jenny s.n. 6.1.1991 (BSB) Argentina 605 — AF396906 Heliotropium angiospermum Murray Hilger Cuba_99/44 (BSB) Cuba 454 — AF396907 Heliotropium antillanum Urb. Hilger Cuba_99/26 (BSB) Cuba 442 AY376175 AF396891 Heliotropium arbainense Fresen. Förther 4049 (BSB) Egypt 606 AY376176 AF396916 Heliotropium arborescens L. commercial cultivated plant (BSB) not indicated 563 AY376177 AF396896 INGROUP – Heliotropiaceae Heliotropium asperrimum R.Br. Craven 9671 (CANB) Australia 842 AY376178 AF402586 Heliotropium bacciferum Forssk. Podlech 35182 (M) Algeria 115 AY376179 AY377793 Heliotropium ballii Domin Craven 8573 (M) Australia 741 — Heliotropium bursiferum Wr. ex Griseb. Hilger Cuba_99/25 (BSB) Cuba 443 AY376180 AF396888 Heliotropium campestre Griseb. Jenny 37 (BSB) Argentina 207 AY376181 AF396887 Heliotropium chrysanthum Phil. Hilger Arg_95/81 (BSB) Argentina 20 AY376182 — Hilger Arg_95/92 (BSB) Argentina 515 — Hilger Nam_93/10 (BSB) Namibia 10B AY376183 AY377795 Heliotropium ciliatum Kaplan AY377794 AF396894 22 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Species Voucher Source BSB GenBank accession accession number number trnL GenBank accession number ITS1 Heliotropium confertiflorum Boiss. & Noe Akhani & Lari 5591 (KAS) Iran Heliotropium convolvulaceum (Nutt.) A.Gray Hilger USA_92/77 (BSB) USA: California 1042 — Miller 6691 (BSB) USA: Colorado 1043 AY376187 AY377797 Heliotropium cupressinum Craven Fryxell & Craven 4006 (M) Australia 134 — Heliotropium curassavicum L. var. argentinum I.M.Johnst. Hilger Arg_95/82 (BSB) Argentina 232 AY376185 AF396898 600 AY376184 AY377798 AY377796 AY377799 subsp. oculatum (A.Heller) Thorne Hilger USA_94/21 (BSB) USA 3B AY376186 AF396897 Heliotropium digynum (Forrsk.) Asch. ex C.Chr. Hilger Israel_94/23 (BSB) Israel 211 AY376188 AF396915 Heliotropium elongatum (Lehm.) I.M.Johnst. Hilger Arg_95/15 (BSB) Argentina 966 — Heliotropium erosum Lehm. Zippel 2000/69 (BSB) Spain: Tenerife 677 AY376189 AY377801 AY377800 Heliotropium europaeum L. Hilger Bg_97/6 (BSB) Bulgaria 496, 581 AY376193 AF402587 Heliotropium giessii Friedr.-Holzh. Hilger Nam_93/3 (BSB) Namibia 607 AY376194 AF396917 Heliotropium hirsutissimum Grauer Nogatz s.n. 28.09.1997 (BSB) Turkey 580 AY376190 — Kagiampaki s.n. 24.07.2000 (BSB) Greece: Crete 692 — Heliotropium humifusum Kunth Löschner s. n. March 2000 (BSB) Cuba 673 AY376191 AF396890 Heliotropium incanum Ruiz & Pav. Weigend 2000/162 (M) Peru 733 AY376192 AY176077 Heliotropium indicum L. Hilger Cuba_22/99 (BSB) Cuba 448 — Heliotropium krauseanum Fedde Weigend & Förther 97/727 (M) Peru 233 AY376195 AF396909 Heliotropium kurtzii Gangui Weigend et al. 5914 (BSB) Argentina 1066 — AY377803 Heliotropium linariaefolium Phil. Dillon et al. 5502 (M) Chile 713 — AY377804 Namibia Heliotropium lineare (A.DC.) Gürke Hilger Nam_ 93/7 (BSB) AF396912 AY377802 8B AY376196 AY377805 Heliotropium mandonii I.M.Johnst. Weigend, cult. Botanical Garden Ecuador of München-Nymphenburg, Germany 1.9.1997 (BSB) 205 AY376197 AF396895 Heliotropium mendocinum Phil. Hilger Arg_95/77 (BSB) Argentina 235 AY376198 AF396893 Heliotropium microstachyum Ruiz & Pav. Hilger Arg_95/54 (BSB) Argentina 65 AY376199 — Weigend et. al. 97/320 (BSB) Peru 690 — Hilger Nam_93/6 (BSB) Namibia 518, 7B AY376200 AY377806 Heliotropium nelsonii C.H.Wright Heliotropium nicotianaefolium Poir. Hilger Arg_95/56 (BSB) Hilger Arg_s.n. 28.01.1998 (BSB) AF396908 Argentina 13H AY376201 — Argentina 720 — AY377807 23 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Species Voucher Source BSB GenBank accession accession number number trnL GenBank accession number ITS1 Heliotropium oliverianum Schinz Hilger Nam_93/16 (BSB) Namibia 608, 15B AY376202 AF396913 Heliotropium ovalifolium Forssk. Hilger Nam_93/5 (BSB) Namibia 12B AY376203 AF396886 Heliotropium paronychioides A.DC. Hilger Arg_95/96 (BSB) Argentina 23H AY376204 — Grau 2750 (M) Chile 709 — Heliotropium patagonicum (Speg.) I.M.Johnst. Weigend et al. 5940 (BSB) Argentina 1062 AY376205 AY377809 Heliotropium pinnatisectum R.L.Pérez-Mor. Weigend et al. 5901 (BSB) Argentina 1060 AY376206 AY377810 AY377808 Heliotropium procumbens Mill. Feuerer 9452b (BSB) Bolivia 210 AY376207 AF396885 Heliotropium pulvinum Craven Craven 8583 (M) Australia 132 — Heliotropium pycnophyllum Phil. Dillon & Dillon 6041 (M) Chile 714 AY376208 AY377812 Heliotropium rariflorum Stocks subsp. hereroense (Schinz) Verdc. Hilger Nam_93/23 (BSB) Namibia 16B AY376209 AF396889 Heliotropium stenophyllum Hook. & Arn. Dillon 5428 (M) Chile 715 — AY377813 Heliotropium strigosum Willd. Müller 564-b (FR) West Africa 1171 — AY377814 Heliotropium styotrichium Craven Craven 9687 (CANB) Australia 843 AY376212 AY377815 Heliotropium suaveolens M.Bieb. Hilger Bg_97/5 (BSB) Bulgaria 204 AY376210 AF396911 Heliotropium supinum L. Hilger s.n. anno 1985 (BSB) Italy 350 AY376211 AF396919 Heliotropium taltalense (Phil.) I.M.Johnst. Dillon & Teillier 5233 (M) Chile 716 — Heliotropium tenuifolium R.Br. Craven 9688 (CANB) Australia 844 AY376213 AY176079 Heliotropium transalpinum Vell. Hilger Arg_95/23 (BSB) Argentina 611 — Heliotropium tubulosum DC. Hilger Nam_93/18 (BSB) Namibia 208, 13B AY376214 AY377817 Heliotropium veronicifolium Griseb. Hilger Arg_95/20 (BSB), cult. Botanical Garden of MünchenNymphenburg, Germany Argentina 206 AY376215 AY377818 Heliotropium zeylanicum (Burm.f.) Lam. Hilger Kenya_94/4 (BSB) Kenya 612 AY376216 AY377819 Hilgeria hypogaea (Urban & Eckman) Förther Ekman s.n. 1927 (Isotypus) (M) Cuba 895 AY376217 — Briggs 226 (M) Cuba 894 — AY377820 Hilgeria serpylloides (Griseb.) Förther Webster 3966 (M) Cuba 896 — AY377821 Ixorhea tschudiana Fenzl cult. Botanical Garden of München-Nymphenburg, Germany (BSB) Argentina 176 AY376218 AF396880 AY377811 AY377816 AF396904 24 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Species Voucher Source BSB GenBank accession accession number number trnL Nogalia drepanophyllum (Baker) Verdc. Kilian 6603 (BSB) GenBank accession number ITS1 Yemen 799 AY376219 AY377822 Schleidenia baclei DC. var. rostratum Gilges 685 (M) I.M.Johnst. Zambia 707 AY376220 AY377823 Schleidenia lagoensis Warm. Schessl 2831 (M) Brazil 708 AY376221 AF396892 Tournefortia argentea L.f. Tillich 3555, cult. Botanical Garden of MünchenNymphenburg, Germany Mauritius 319, 687 AY376222 AF396900 Tournefortia breviloba Krause Weigend & Horn 3827 (BSB) Ecuador 823 AY376223 AY377824 Tournefortia chinchensis Killip Weigend et al. 2001/160 (BSB) Peru 949 AY376224 AY377825 Tournefortia fuliginosa Kunth Weigend & Horn 3834 (BSB) Ecuador 825 AY376225 AY377826 Tournefortia gnaphalodes (L.) Kunth Hilger Cuba_99/34 (BSB) Cuba 441 AY376226 AF396903 Tournefortia hirsutissima L. Stenzel 96/32 (BSB) Cuba 74 AY376227 AF396901 Tournefortia luzonica I.M.Johnst. Liede 3302 (BSB) Philippines 601, 430 AY376228 AF396899 Tournefortia microcalyx (Ruiz & Pav.) I.M.Johnst. Weigend & Dostert 97/5 (M) Peru 686 — Tournefortia polystachya Ruiz & Pav. Weigend & Horn 3869 (BSB) Ecuador 822 AY376229 AY377827 Tournefortia psilostachya Kunth Weigend & Weigend 2000/339 (M) Peru 718 — AF396883 Tournefortia rollotii Killip Schnetterer s.n. 23.02.1983 (BSB) Colombia 150 — AY377828 Tournefortia salzmannii DC. Franca & Melo 16843 (M) Brazil 719 — AF396884 Tournefortia tarmensis (Krause) Macbride Weigend et al. 2001/19 (BSB) Peru 946 AY376230 AY377829 Tournefortia ternifolia Kunth Weigend et al. 2001/25 (BSB) Peru 947 AY376231 AY377830 Tournefortia undulata Benth. Weigend & Förther 1997/880 (BSB) Peru 824 AY376232 AY377831 Tournefortia usambarensis (Verdc.) Verdc. Lovett & Thomas 2464 (MO) Tanzania 794 — AY176083 Tournefortia cf. virgata Ruiz & Pav. Weigend et al. 2001/48 (BSB) Peru 950 — AY377832 Tournefortia volubilis L. Hilger Mex_1980/6 (BSB) Mexico 147 AY376233 AF396882 cult. Botanical Garden of München-Nymphenburg, Germany not indicated 689 AY376234 AF396881 AF396905 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 25 Phylogenetic analysis Sequences were edited and manually aligned with the Alignment-Editor Align 32 (HEPPERLE 1997). The alignment of the ITS1 region was improved by using the secondary structure according GOTTSCHLING et al. (2001). “Hairpin” and “stem loop” regions were identified and separately aligned. Indels were coded as “missing characters”; parsimony informative indels were coded separately as present/absent following the “simple gap coding” method of SIMMONS & OCHOTERENA (2000). Indels which are present or absent only in different accessions of the same species were not coded. Indels of doubtful homology were not coded (especially in overlapping gap regions, which occur preferable in helix I). All alignments are available from the authors on request. Phylogeny analyses were performed by PAUP* 4.0b1 (PC version) and TREECON for Windows (version 1.3b). Parsimony analyses (PAUP: SWOFFORD 2002) were performed using a heuristic search. The starting trees were obtained by random stepwise addition to the taxa with 1000 replicates, tree-bisection-reconnection (TBR) branch swapping, saving all parsimonious trees (“MulTrees” on), and MAXTREES set to “autoincrease”. First, starting trees were obtained (addition sequence random, 1000 replicates, TBR branch swapping, “MulTrees” off, “steepest descent” on). The resulting shortest starting trees were then subjected to TBR branch swapping (“MulTrees” on) to the limit of computer capacity. All characters were weighted equally, and character state transitions were treated as unordered. Bootstrap resampling (FELSENSTEIN 1985) was performed with 1000 replicates and a heuristic search, with random addition of taxa (10 addition sequence replicates), with a limit of 100 trees kept at each step. In addition, Neighbor-Joining analyses (SAITOU & NEI 1987) were performed using a heuristic search run in TREECON (VAN DE PEER & DE WACHTER 1994). Gaps were treated as missing data instead of “gap coding”. Sequence divergence values were calculated by KIMURA ’s two-parameter distance models (K IMURA 1980) and a bootstrap analysis with 1000 replicates. The trnL dataset was separately analyzed, followed by a combined trnL and ITS1 dataset to find the robust main clades. Each main clade was then individually analyzed using ITS1 sequences. With regard to the analysis of the HELIOTROPIUM II clade we combined the trnL and ITS1 datasets, because in this case resolution increased. The number of outgroup species was reduced in order to minimize the homoplasy content. The names of the main clades identified in this study are indicated in capital letters. Results 1. Complete DNA sequence analysis trnL dataset — Analysis of the trnL dataset, including 20 separated coding indels (of a total of 568 characters 106 are parsimony informative), resulted in 40,100 most parsimonious trees (l=207 steps, CI=0.899, RI=0.975, HI=0.101; strict consensus tree see Fig. 1). With respect to the outgroup, all ingroup taxa constitute a monophylum (99% bootstrap support). Within the strict consensus tree, two main clades with high bootstrap values can be distinguished. In the first large clade (77% bootstrap support), IXORHEA (Ixorhea) is sister (99% bootstrap support) to MYRIOPUS (Tournefortia section Cyphocyema I.M.Johnst.) and EUPLOCA (Heliotropium section Orthostachys R.Br., Schleidenia, Hilgeria), the latter supported by 100% each. The second large clade (100% bootstrap support) comprises all remaining species of Heliotropiaceae 26 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 1. Strict consensus tree of the analyzed species of Heliotropiaceae, computed from the 40,100 most parsimonious trees of the trnL dataset (l=207 steps, CI=0.899, RI=0.975, HI=0.101). Important clades are indicated. The numbers above the branches are bootstrap percentages. Percentages <50% are not shown. H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 27 and is subdivided into three subclades. Of these, HELIOTHAMNUS (Heliotropium section Heliothamnus I.M.Johnst.) forms the unsupported sister group of the remaining species (with 66% bootstrap value weakly supported). A second, well-supported subclade (80% bootstrap support), comprises the Heliotropium species of the Old World, including Ceballosia, Nogalia, and Argusia sogdiana. Together with the unsupported Argusia sibirica we call it HELIOTROPIUM II. All Heliotropium species of the New World and Tournefortia section Tournefortia I.M.Johnst. remain unresolved and form the third subclade, HELIOTROPIUM I. The corresponding KIMURA -2-parameter based neighbor-joining (NJ) tree (Fig. 2) is highly congruent with the parsimony strict consensus tree (Fig. 1). It shares nearly the same topology, and revealed identical clades. On clade one IXORHEA, MYRIOPUS, and EUPLOCA are less well-supported (58% bootstrap support). On clade two HELIOTHAMNUS is also separated. HELIOTROPIUM I (59% bootstrap support) constitutes the sister group of HELIOTROPIUM II (62% bootstrap support). Combined trnL-ITS1 dataset — Analysis of the combined trnL-ITS1 dataset, including 20 separated indels of the trnL dataset whereas ITS1 remains uncoded, did not change the well-supported clades of the single analysis of the trnL dataset. The combined analysis (of a total of 912 characters 251 are parsimony informative) resulted in 2,604 most parsimonious trees (l=950 steps, CI=0.622, RI=0.832, HI=0.378; strict consensus tree see Fig. 3). The ITS1 dataset within Heliotropiaceae is very heterogenous. Nevertheless, bootstrap values of the combined analysis partly increase in the main clades which are retrieved in the single marker analysis. IXORHEA is isolated in combined analysis, due to problematic positions in the ITS1 alignment. This leads to a lower bootstrap support (75%) for the remaining species of Heliotropiaceae. However, the first large clade (83% bootstrap support) of combined analysis contains MYRIOPUS and the large EUPLOCA crown clade as sisters with 100% bootstrap support each. The second large clade with all other species of Heliotropiaceae is still supported by 100%. HELIOTHAMNUS (99% bootstrap support) is basal situated to HELIOTROPIUM I and II (72% bootstrap support). HELIOTROPIUM I is supported by 66%, whereas HELIOTROPIUM II is partly unresolved. The corresponding KIMURA NJ tree (Fig. 4) shares the same topology regarding the position of IXORHEA, and the two large and well-supported main clades (92% respectively 97% bootstrap support). Within the large HELIOTROPIUM and HELIOTHAMNUS clades tree resolution collapses, due to increasing homoplasy content of the ITS1 dataset within the entire family, and due to the lack of “gap coding” which is in the programs used for the Neighbor-Joining analysis impossible. 28 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 2. Neighbor-joining tree of the analyzed species of Heliotropiaceae of the trnL dataset. Important clades are indicated. The numbers above the branches are bootstrap percentages (1000 replicates, percentages <50% are not shown, scale bar 2% distance). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 29 Fig. 3. Strict consensus tree of the analyzed species of Heliotropiaceae, computed from the 2,604 most parsimonious trees of the combined trnL and ITS1 datasets (l=950 steps, CI=0.622, RI=0.832, HI=0.378). Important clades are indicated. The numbers above the branches are bootstrap percentages. Percentages <50% are not shown. 30 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 4. Neighbor-joining tree of the analyzed species of Heliotropiaceae of the combined trnL and ITS1 dataset. Important clades are indicated. The numbers above the branches are bootstrap percentages (1000 replicates, percentages <50% are not shown, scale bar 5% distance). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 31 2. ITS1 trees of main clades identified in 1. The ITS1 dataset of the main clades EUPLOCA-MYRIOPUS, HELIOTROPIUM I, and HELIOTROPIUM II were separately analyzed, in order to obtain trees with increased resolution. Additional species were added. MYRIOPUS and EUPLOCA — Analysis of the ITS1 dataset of the MYRIOPUS and EUPLOCA clade, including 7 separated coding indels (of a total of 309 characters 107 are parsimony informative), resulted in 112 most parsimonious trees (l=332 steps; CI=0.723, RI=0.803, HI=0.277; majority-rule consensus tree see Fig. 5a). Well-supported clades are MYRIOPUS (100% bootstrap support) and EUPLOCA (96% bootstrap support). They occur as a sister group relationship, whereas IXORHEA does not cluster and fall into the outgroup. Within EUPLOCA, the South American species Heliotropium chrysanthum and H. mendocinum, characterized by underground tubers, form a strongly supported clade (99% bootstrap support, “tuber” clade) and are, weakly supported (69% bootstrap support), sister to the remaining species of EUPLOCA. The latter fall into five subclades, three of them are well-supported. The relationships between these subclades are unresolved. The first subclade (90% bootstrap support) corresponds to Heliotropium sect. Orthostachys subsect. Ebracteata I.M.Johnst. (South American H. campestre and H. procumbens, African H. ovalifolium). North American H. convolvulaceum is unsupported in sister group relationship to an unresolved subclade comprising: The Caribbean clade (Caribbean species Heliotropium humifusum, H. bursiferum, and Hilgeria), the Schleidenia clade (New and Old World species of the genus Schleidenia), and the African-Australian species. In this subclade, the Caribbean clade (including the genus Hilgeria) is strongly supported (90% bootstrap support) as well as the African-Australian subclade (95% bootstrap support). The Schleidenia subclade remains unsupported. The Neighbor-Joining analysis (Fig. 5b) shows nearly the identical topology. Long distances and high bootstrap percentages characterize the main clades and subclades: MYRIOPUS, EUPLOCA, “Ebracteata” clade, “tuber” clade, “Caribbean” clade, and “African-Australian” clade. The major deviation from the parsimony analysis is the position of the Ebracteata clade (97% bootstrap support), which constitute in the NJ analysis unsupported sister to the remaining species of EUPLOCA. The three sequenced species of the genus Schleidenia (Heliotropium antillanum, Schleidenia baclei, and S. lagoensis) are in unresolved position between the Caribbean and African species. HELIOTROPIUM I — Analysis of the ITS1 dataset of the HELIOTROPIUM I clade, including 6 separated coding indels (of a total of 317 characters 99 are parsimony informative), resulted in 4,317 most parsimonious trees (l=460 steps; CI=0.724, RI=0.743, HI=0.276; majority-rule consensus tree see Fig. 6a). The Old World representatives (HELIOTROPIUM II, excluding 32 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 5. a) Majority-rule consensus tree of the analyzed species of the MYRIOPUS and EUPLOCA clade, computed from the 112 most parsimonious trees of the ITS1 dataset (l=332 steps, CI=0.723, RI=0.803, HI=0.277). Important clades are indicated. The numbers above the branches are bootstrap percentages. Percentages 2% are not shown. The numbers below the branches are majority percentages <100%. b) Corresponding neighbor-joining tree. Important clades are indicated. The numbers above the branches are bootstrap percentages (1000 replicates, percentages <50% are not shown, scale bar 10% distance). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 33 Ceballosia) constitute, weakly supported (64% bootstrap support), a sister group to HELIOTROPIUM I. Ceballosia is related to HELIOTROPIUM I, but unsupported. Within HELIOTROPIUM I nine subclades are distinguishable, but mostly in unsupported relationships. Within HELIOTROPIUM I, H. sect. Cochranea (Miers) Kuntze (84% bootstrap support) is undoubtedly basal to the remaining species of HELIOTROPIUM I (73% bootstrap support). These remaining species cluster in various subclades of the sections Plagiomeris I.M.Johnst. (100% bootstrap support), Tiaridium (Lehm.) Griseb. (99% bootstrap support), Heliotrophytum G.Don. (100% bootstrap support), Hypsogenia I.M.Johnst., Coeloma (DC.) I.M.Johnst. (96% bootstrap support), and Schobera (Scop.) I.M.Johnst. One unsupported subclade comprises the “halophytes” [species of H. sect. Platygyne Benth. (99% bootstrap support), including Tournefortia argentea and T. gnaphalodes]. Another unsupported subclade comprises the remaining species of Tournefortia section Tournefortia (“Tournefortia” clade). The Neighbor-Joining analysis (Fig. 6b) results, with respect to the main clades, in the same topology of the tree like the parsimony analysis. HELIOTROPIUM I (56% bootstrap support), including Ceballosia, is in sister group relationship to HELIOTROPIUM II (69% bootstrap support). Section Cochranea (98% bootstrap support) is the sister group of the remaining HELIOTROPIUM I (74% bootstrap support) species, which cluster in the same subclades as in the parsimony analysis. The exceptions are Tournefortia argentea and T. gnaphalodes, both clustering randomly. The relationships between the subclades remain unsupported. They do not agree with the results obtained from the majority-rule consensus tree (Fig. 6a). Combined trnL-ITS1 dataset of HELIOTROPIUM II — Analysis of the combined trnL and ITS1 datasets of the HELIOTROPIUM II clade, including 7 separated coding indels (of a total of 859 characters 100 are parsimony informative), resulted in 84 most parsimonious trees (l=373 steps; CI=0.751, RI=0.748, HI=0.249; majority-rule consensus tree see Fig. 7a). Supported by 70% bootstrap value, the HELIOTROPIUM I representatives, including Ceballosia, constitute an unsupported sister group to HELIOTROPIUM II (excluding Ceballosia). Within HELIOTROPIUM II five subclades are identified, three of them supported by high bootstrap values. A well-supported (95% bootstrap support) large crown clade comprises species of the H. sect. Heliotropium (= Heliotropium L. sects. Agoraea Bunge, Gyrostachys G.Don), Pleurolasia Bunge, Odontotropium Griseb., and Chamaetropium Griseb. Heliotropium supinum (sect. Chamaetropium) clusters well with Nogalia drepanophyllum . The sister group (99% bootstrap support) to this crown clade constitute the species of H. sect. Pterotropium (DC.) Bunge. The remaining species of HELIOTROPIUM II belong to the sections Rutidotheca (A.DC.) Verdc., Zeylanica Förther, Pseudocoeloma Förther and the genus Argusia, and they remain all in unsupported basal position of the total HELIOTROPIUM II clade. 34 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 6. a) Majority-rule consensus tree of the analyzed species of the HELIOTROPIUM I clade, computed from the 4,317 most parsimonious trees of the ITS1 dataset (l=460 steps, CI=0.724, RI=0.743, HI=0.276). Important clades are indicated. The numbers above the branches are bootstrap percentages. Percentages <50% are not shown. The numbers below the branches are majority percentages <100%. A higher resolution of the dataset is found in the Neighbor-Joining Analysis (Fig. 7b). General topology is identical to the parsimony analysis. The Heliotropium supinum / Nogalia drepanophyllum clade constitutes the well-supported (74% bootstrap support) sister group of the Heliotropium / Pleurolasia / Odototropium crown clade. The basal clades (Heliotropium sections Rutidotheca, Zeylanica, Pseudocoeloma, and genus Argusia) of HELIOTROPIUM II show short distances between each other and long distances within the taxa of each clade. ITS1 in the region of helix 1 — The main characteristic of all HELIOTROPIUM II species, except the basally situated Argusia sibirica and Ceballosia fruticosa, is a radical abridgement of helix I (about secondary structure of Heliotropiaceae see Figs. 6 and 8 in GOTTSCHLING et al. 2001), which results in a large deletion in the alignment (Fig. 8). All species of Heliotropiaceae, except HELIOTROPIUM II species (exclude Ceballosia fruticosa and Argusia sibirica), H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 35 Fig. 6. b) Corresponding neighbor-joining tree of the analyzed species of the HELIOTROPIUM I clade. Important clades are indicated. The numbers above the branches are bootstrap percentages (1000 replicates, percentages <50% are not shown, scale bar 10% distance). are characterized by a long helix I of pairing regions, about up to 25 bp (Fig. 9a, b, d; the homologous regions are labelled I to III), or up to 13 bp long (Fig. 9c). A partial loss of the apical region of helix I (Fig. 9c; loss of homologous region III) was found in EUPLOCA, HELIOTROPIUM I and is characteristic for 36 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 7. a) Majority-rule consensus tree of the analyzed species of the HELIOTROPIUM II clade, computed from the 84 most parsimonious trees of thecombined trnL and ITS1 datasets (l=373 steps, CI=0.751, RI=0.748, HI=0.249). Important clades are indicated. The numbers above the branches are bootstrap percentages. Percentages <50% are not shown. The numbers below the branches are majority percentages <100%. b) Corresponding neighbor-joining tree. Important clades are indicated. The numbers above the branches are bootstrap percentages (1000 replicates, percentages <50% are not shown, scale bar 10% distance). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 37 Fig. 8. Examples of proposed alignment of helix I, considering secondary structure of the ITS1. Pairing bases are indicated bold. Partial abridgements of helix I are highlighted. 38 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 9. Examples of helix I of ITS1 transcript secondary structure. Homologous regions are labelled I to III. HELIOTHAMNUS (Fig. 8). A nearly complete abridgement (Fig. 9e; loss of homologous regions II, and III) found only in HELIOTROPIUM II for the most species (Fig. 8). A total abridgement of helix I found within HELIOTROPIUM II for Argusia sogdiana, Heliotropium nelsonii, and H. zeylanicum (Fig. 9f). The region of helix I is very informative, but it is difficult to implement a “gap coding” here. Due to completely overlapping gaps, it is not possible to detect whether a subset of bases, lost before the indels, is responsible for the longer gap. In the region of helix I we therefore prefer not to use “gap coding”. This method results in a decrease of bootstrap support, because large informative regions of ITS1 sequences were not used for the phylogenetic sequence analysis. H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 39 Discussion Intrafamilial relationships These molecular studies of Heliotropiaceae strongly support the results of DIANE et al. (2002a), which contradicted traditional taxonomic circumscription particularly with regard to the large core-genera Heliotropium and Tournefortia (CANDOLLE 1845, GÜRKE 1893, JOHNSTON 1928, 1930, 1935a, FÖRTHER 1998). The inclusion of a second marker (the trnLUAA intron) besides the ITS1 region in our analyses led to better differentiation of main clades and higher resolution within each of them. A summary of the results including morphological-anatomical traits is shown in Fig. 10. Our results show that each of the two clades, IXORHEA, MYRIOPUS and EUPLOCA on the one hand and HELIOTHAMNUS, HELIOTROPIUM I, and HELIOTROPIUM II on the other hand, constitute a monophyletic group. Both clades are well-supported by both the trnL and the combined trnL/ITS1 dataset. In the combined trnL/ITS1 dataset only the position of IXORHEA remains unresolved. In the following, each main clade will be specified. IXORHEA Analysis of ITS1 sequences could not unambiguously resolve the exact systematic position of monotypic Ixorhea (DIANE et al. 2002a). In contrast, the more conservative sequences of trnL clarify its sister group relationship to the MYRIOPUS / EUPLOCA clade. Ixorhea is characterized mainly by autapomorphic morphological traits: elongated and winged mericarpids enclosing a straight embryo, and the whole plant being completely resinous (DIFULVIO 1978). However, independent of its exact systematic position, Ixorhea does not cluster inside any clade. Therefore, with regard to nomenclature, no changes are suggested. MYRIOPUS Without doubt the species of Tournefortia sect. Cyphocyema, clustering in the first main clade, are not closely related to Tournefortia sect. Tournefortia, appearing in the second main clade. Traditionally Tournefortia species are defined mainly by obviously convergent traits such as liana or subscandent habit, or the presence of drupaceous fruits. The assumption of DIANE et al. (2002a) that MYRIOPUS species take a basal position within Heliotropiaceae must be revised. All available molecular data speak for the well-supported sister group relationship of MYRIOPUS and EUPLOCA. Such a close relationship had 40 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Fig. 10. Phylogeny of Heliotropiaceae based on molecular data, including morphological-anatomical traits. H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 41 already been assumed by JOHNSTON (1930), but without explanation. The following morphological-anatomical apomorphies support this assumption: a curved embryo, corolla lobes with involute margins (DIANE et al. in press, figs. 1B, I), and the complete lack of calcium oxalate druses in leaf mesophyll. Instead of crystal druses, bundles of calcium oxalate needles are present in the epidermis in some species of MYRIOPUS and EUPLOCA (DIANE et al. 2003). The characteristic papillose to pubescent apex of the connate anthers, which close the corolla tube above the style-stigma-complex, seems to be plesiomorphic within Heliotropiaceae. Beside MYRIOPUS and EUPLOCA these characters also occur within Ixorhea and the HELIOTHAMNUS clade. However, in Ixorhea the anthers are merely long protracted and apically compressed, and in HELIOTHAMNUS the pubescent apices of the anthers are only sometimes connected. The monophyly of the MYRIOPUS clade, as well as the clear morphological circumscription justify the general acceptance of the genus Myriopus Small (SMALL 1933). The species of MYRIOPUS are characterized by the following traits: lianas with drupaceous, deeply 4-lobed fruits with one-seeded and 4-layered endocarpids (DIANE et al. in press, fig. 1D), a thick tissue of transfer cells in the placenta region (DIANE et al. 2002b, fig. 14-16), characteristic flowers with subulate corolla lobes, inflated bases of the corolla tubes, and involute corolla margins in the buds. The necessary new combinations, of the species investigated, from Tournefortia to Myriopus are presented in Appendix 1. EUPLOCA The species of Heliotropium section Orthostachys are distinctly separated from the remaining species of Heliotropium and cluster together with Hilgeria and Schleidenia. The EUPLOCA clade shows a higher affinity to the genus Myriopus than to the remaining species of Heliotropium. This relationship is also well-supported by morphological-anatomical data. All species of EUPLOCA show mericarpid or endocarpid structures with surface sculpturings described as “pits” (precise description in DIANE et al. 2002a), kranz-chlorenchyma organisation in leaves of almost all species (exceptions found only in Heliotropium sect. Ebracteata, DIANE et al. 2003), and the exclusive occurrence of characteristic trichomes on a pedestal of distinctly enlarged foliar epidermis cells (trichome type 3 in DIANE et al. 2003). Within the EUPLOCA clade we identified several subclades. One well-supported subclade comprises South American species (Heliotropium chrysanthum, H. mendocinum) with underground tubers as a particular adaptation to semi-arid habitats with seasonal or erratic dry periods. Heliotropium subsect. Ebracteata (as Ebracteata-clade), appears to be monophyletic. The species are characterized by completely bractless inflorescences, whereas the remaining species of EU- 42 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae PLOCA are bracteate. The Ebracteata-clade comprises Old World (Heliotropium ovalifolium) and New World (H. procumbens, H. campestre) species as well as few species with kranz-chlorenchyma organisation (H. campestre). However, most species are lacking kranz-chlorenchyma organisation (H. procumbens, H. ovalifolium) in the leaves (DIANE et al. 2003, FROHLICH 1978). Due to the contradictory position in parsimony or NJ trees, the exact phylogenetic position of the Ebracteata clade within EUPLOCA remains unclear. Furthermore, we identified a Caribbean clade, which comprises dwarf-shrubs with multiflowered inflorescences (Heliotropium bursiferum), procumbent subshrubs with solitary flowers (H. humifusum), and procumbent herbs with solitary flowers and postflorally elongating pedicels (defined as the genus Hilgeria). The species of the pantropical genus Schleidenia clustered unsupported between the Caribbean and African-Australian clade. Nevertheless, morphological traits, in particular the characteristic of drupaceous fruits (FÖRTHER 1998), indicate this group as a monophylum. The African-Australian clade comprises in basal positions the species Heliotropium rariflorum (distributed from southeastern Africa, the Arabian Peninsula and southern Iran to southern Pakistan) and H. strigosum (from West Africa, the Arabian Peninsula to India, Pakistan, and Australia) and in crown position a large number of Australian species. H. strigosum is sister to an apparently evolutionary young Australian group which undertook a rapid radiation while colonizing this continent (CRAVEN 1996). Summarizing all urges a formal taxonomic recombination of EUPLOCA, including all species of Heliotropium section Orthostachys, Schleidenia, and Hilgeria into the genus Euploca Nutt. (NUTTALL 1837) the oldest available generic name in this group. Schleidenia Endl. (ENDLICHER 1839) as formerly proposed (DIANE et al. 2002a) is younger. Combinations are presented in Appendix 1. HELIOTHAMNUS, HELIOTROPIUM I and II These clades constitute a monophylum, well-supported by both molecular and morphological (presence of always straight embryos) data. Within this large clade, comprising species of Heliotropium, Tournefortia sect. Tournefortia, Ceballosia, Argusia and Nogalia, not all systematic relationships between the clades are undoubtedly clarified. HELIOTHAMNUS The species of the Andean Heliotropium sect. Heliothamnus are excluded as a well-supported clade from HELIOTROPIUM I and II. This clade, probably H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 43 segregated with the upfolding of the Andes, is characterized by plesiomorphic traits such as one-seeded mericarpids and anthers with pubescent and sometimes connected apices. HELIOTROPIUM I and II HELIOTROPIUM I and HELIOTROPIUM II are well-supported sister clades. They differ morphologically from the remaining species of Heliotropiaceae by the lack of pubescent or papillose apices of the anthers, which are never connate. Two-seeded endocarpids, and “empty chambers” are present only in these two groups. Obvious air-filled empty chambers next to the locules developed several times independently. They are of different shape and localization and are present in species of different clades [see figures of e.g. Tournefortia usambarensis (“Tournefortia” clade of HELIOTROPIUM I) in VERDCOURT 1991: 49 fig.10; Ceballosia fruticosa (HELIOTROPIUM II) in HILGER 1989: 127 fig. 6d; Heliotropium indicum (Tiaridium clade of HELIOTROPIUM I) in ROSANOFF 1866: plate VI, fig. 15, 18, 19]. Within HELIOTROPIUM I empty chambers are present in all two-seeded “Tournefortia” species, within Heliotropium sect. Heliotrophytum only in H. nicotianaefolium, and within sect. Tiaridium only in H. indicum. HELIOTROPIUM I This clade comprises Heliotropium species distributed in the New World and the species of Tournefortia sect. Tournefortia as a part of Heliotropium. With respect to the sectional relationships of Heliotropium species in HELIOTROPIUM I they cluster in by FÖRTHER (1998) accepted sections, but with unresolved relationships among one another. The only exceptions are: a) the species of H. sect. Cochranea. They are well-supported in a basal position within HELIOTROPIUM I. b) all halophytic species. They comprise species of Heliotropium sect. Platygyne, Tournefortia gnaphalodes, and T. argentea and cluster together. The systematic position of both Tournefortia species has been controversially discussed till now. In Table 2 we show the nomenclatural synopsis of these (and other Heliotropiaceae) species. JOHNSTON (1930, 1935a, 1949, 1951), placed these Tournefortia species several times either in Messerschmidia or Tournefortia, and pointed out that these species are closer related to Heliotropium than to Tournefortia. Our results demonstrate that the halophytes constitute a distinct subclade of HELIOTROPIUM I. The most important and surprising result, pointed out for the first time in DIANE et al. (2002a), is the unrecognized fact that the species of Tournefortia Heliotropium Tournefortia Messerschmidia Argusia argentea (L.f.) I.M.Johnst., argentea (L.f.) H.Heine, Fl. J. Arnold Arbor. 16: 164. 1935. N. Caled. & Depend. 7: 109. 1976. gnaphalodes L., Syst. Nat. ed. 10 2: 913. 1759. gnaphalodes (L.) R.Br., Prod.: 496. 1810. gnaphalodes (L.) I.M.Johnst., J. Arnold Arbor. 16: 165. 1935. japonicum A.Gray, Mem. Amer. Acad Arts, n.s., 6: 403. 1859. in FÖRTHER (1998: 201) and JOHNSTON (1935a: 164) sibirica L., Sp. Pl.: 141. 1753. sibirica var. angustior (DC.) sibirica (L.) Dandy, Bot. J. W.T.Wang, Fl. Reipubl. Popul. Linn. Soc. 65: 256. 1972. Sin. 64(2): 34. 1989. sibirica var. angustior (DC.) G.L.Chu & M.G.Gilbert, Novon 5: 17. 1995. sogdianum Bunge, Beitr. Kenntn. Fl. Russl.: 227. 1852. sogdiana (Bunge) Popov, in sogdiana (Bunge) Riedl, in Bull. Sect. Turkest. Soc. Russe K.H. Rechinger, Fl. Iran., Geogr. 15: 52. 1922. Lief. 48: 9. 1967. messerschmidioides Kuntze, Revis. Gen. Pl.: 438. 1891. in FÖRTHER (1998: 129) fruticosa (L.f.) Ker., Bot. Reg. 6: 464. 1820. fruticosa L.f., Suppl. Pl.: 132. 1781. drepanophyllum Baker, Bull. Misc. Inform. 93 (art. 407): 336. 1894. For further synonymy see FÖRTHER (1989) and JOHNSTON (1935a, 1935b, 1949, 1951). gnaphalodes (L.) H.Heine, Fl. N. Caled. & Depend. 7: 108. 1976. other genera Mallotonia gnaphalodes (L.) Britton, Ann. Missouri. Bot. Gard. 2: 47. 1915. sogdiana (Bunge) Czerep., Sosud. Rast. SSSR: 112. 1981. Ceballosia fruticosa (L.f.) G.Kunkel ex Förther, Sendtnera 5: 129. 1989. Nogalia drepanophylla (Baker) Verdc., Kew Bull. 43: 432. 1987. H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Heliotropium foertherianum argentea L.f., Suppl. Pl.: 133. Diane & Hilger nom. nov. pro 1781. Tournefortia argentea L.f., Suppl. Pl.: 133. 1781. non Heliotropium argenteum Lehm., Pl. Asperif. Nucif.: 73. 1818. sect. Heliothamnus (FÖRTHER 1998: 80). 44 Tab. 2. Nomenclatural synopsis of Tournefortia argentea, T. gnaphalodes and the species of the genera Argusia, Ceballosia, and Nogalia investigated in this study. Names accepted by FÖRTHER (1989) and used in this study are highlighted. H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 45 section Tournefortia and the Heliotropium species of the New World are very close related. “Tournefortia” represent a lineage of tropical New World Heliotropium species from humid environments (except halophytic T. argentea, T. gnaphalodes), whereas Heliotropium species itself prefer semi-arid habitats. The radiation into humid-tropical conditions caused a change of growth forms and fruit types, and resulted in liana- or subscandent-species with drupaceous fruits. Beside molecular data, a close relationship of “Tournefortia” and New World Heliotropium species is also reflected in leaf anatomy, described in detail by DIANE et al. (2003). Species of both groups are characterized by nearly identical leaf anatomy. They share important characters such as the lack of calcium oxalate tubes in the mesophyll or characteristic unicellular lithocysts with reduced trichome tips, which are not found in the species of the MYRIOPUS or EUPLOCA clades. Thus, the species of Tournefortia section Tournefortia warrant incorporation into the genus Heliotropium. However, before new combinations in the genus Heliotropium are made for “Tournefortia”, the exact relationship within New World Heliotropium needs to be resolved, and a revision of “Tournefortia” is inevitable. Currently, we advocate maintaining the genus Tournefortia, which is easily defined; we conclude that under this definition the genus Heliotropium is paraphyletic. Actually, the only exceptions are halophytic T. gnaphalodes which fall in synonymy of Heliotropium (Tab. 2). For T. argentea we propose a new combination, presented in Appendix 1 (see also Tab. 2). HELIOTROPIUM II This clade comprises Heliotropium species distributed in the Old World plus Argusia sibirica, A. sogdiana, Ceballosia fruticosa, and Nogalia drepanophyllum , which should be maintained in Heliotropium, the genus within which they were described initially (Tab. 2). HELIOTROPIUM II is well-supported by the trnL dataset, and by the loss of nearly the whole helix I in the ITS1 secondary structure. Their very characteristic spear-like trichomes on the leaves (trichome type 5, detailed described in DIANE et al. 2003), support the monophyly of this group of Old World species. Exceptions are Argusia sibirica and Ceballosia fruticosa. Both species take an uncertain intermediate positions between the clades HELIOTROPIUM I and II, supported also by comparative leaf anatomy (DIANE et al. 2003). Within HELIOTROPIUM II three major subclades have been identified. A large subclade is strongly indicated, comprising species of Heliotropium sections Heliotropium, Pleurolasia, and Odontotropium as accepted by FÖRTHER (1998). FÖRTHER (1998) regarded them as closely related because of the shape of the style-stigma-complex and the flower indument. However, our molecular data 46 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae show that none of these three sections is monophyletic. Current morphological circumscriptions do not reflect the phylogenetic relationships; H. arbainense and H. hirsutissimum, two species of sect. Odontotropium, serve as a good example. Section Odontotropium is defined by the occurrence of compact fornices-like intercalary teeth in epipetalous position inside the corolla tube. According to the molecular findings, these traits have to be regarded as convergent, because both species do not cluster together. H. hirsutissimum clusters with H. suaveolens (sect. Heliotropium). Indeed, FÖRTHER (1998) pointed out that H. hirsutissimum and H. suaveolens are nearly similar with the only exception of the lack or presence of intercalary teeth. Therefore, further investigations are necessary to find exact morphological definitions for sectional disposition. In sister group relationship to above mentioned clade is the monotypic section Chamaetropium (H. supinum) which clusters with H. drepanophylla (Tab. 2). The latter separated as Nogalia drepanophyllum by VERDCOURT (1987). Apart from the molecular data, the close relationship of these two species is also supported by morphological traits. Both species exclusively share distinctly urceolate, inflated calyces which enclose the fruits at maturity and act as a dispersing unit. The species of section Pterotropium constitute a natural group which is the sister of the subclades described above. They are characterized by lateral winged or bulging two- or one-seeded fruits (FÖRTHER 1998). The remaining species of HELIOTROPIUM II, H. zeylanicum, H. lineare, H. ciliatum, H. nelsonii, and also Argusia sogdiana, are unsupported in basal position of this clade. The aberrant morphology (FÖRTHER 1998) probably indicates these species as relicts of phylogenetically old lineages. Further investigations are necessary to resolve exact relationships. With regard to both species of the genus Argusia, they do not constitute a natural group, this supported by molecular data and leaf anatomy (DIANE et al. 2003). Appendix 1: Taxonomic recombinations within HELIOTROPIUM I, MYRIOPUS, and EUPLOCA The current available molecular and morphological-anatomical results lead to nomenclatural recombination within the clades HELIOTROPIUM I, MYRIOPUS, and EUPLOCA which are here proposed: HELIOTROPIUM I Heliotropium foertherianum Diane & Hilger nom. nov. º Tournefortia argentea L.f., Suppl. Pl.: 133. 1781 [non Heliotropium argenteum Lehm., Pl. Asperif. Nucif.: 73. 1818, sect. Heliothamnus] (FÖRTHER 1998: 80). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 47 The new name is in honor of HARALD FÖRTHER , the important student of Heliotropiaceae. For synonyms see Tab. 2. MYRIOPUS Myriopus Small, Man. S. E. Fl.: 1131. 1933. º Tournefortia L. sect. Cyphocyema I.M.Johnst., Contr. Gray Herb. 92: 72. 1930. For further synonyms see JOHNSTON (1930). Ty pe : Myriopus volubilis (L.) Small, Man. S. E. Fl.: 1131. 1933. º Tournefortia volubilis L., Sp. Pl.: 140. 1753. For further synonyms see JOHNSTON (1935b, 1949, 1953, 1964). Myriopus psilostachya (Kunth) Diane & Hilger comb. nov. º Tournefortia psilostachya Kunth, in Humboldt, Bonpland & Kunth, Nov. Gen. Sp. 3: 78. 1818. Myriopus salzmannii (DC.) Diane & Hilger comb. nov. º Tournefortia salzmannii DC., Prodr. 9: 524. 1845. For further synonyms see JOHNSTON (1930, 1935b). EUPLOCA Euploca Nutt., Trans. Amer. Phil. Soc., n.s. 5: 189. 1837. º Heliotropium L. sect. Euploca (Nutt.) A.Gray, Proc. Amer. Acad. Arts 10: 49. 1874. = Schleidenia Endl., Gen. Pl.: 646. 1839. = Heliotropium L. sect. Orthostachys R.Br. subsect. Axillaria I.M.Johnst., Contr. Gray Herb. 81: 47. 1928. = Hilgeria Förther, Sendtnera 5: 132. 1989. For further synonyms see FÖRTHER (1998). Ty pe : Euploca convolvulacea Nutt., Trans. Amer. Philos. Soc., n.s. 5: 190. 1837. º Heliotropium convolvulaceum (Nutt.) A.Gray, Mem. Amer. Acad. Arts, n.s. 6: 403. 1859. For further synonyms see FÖRTHER (1998). Euploca antillana (Urb.) Diane & Hilger comb. nov. º Heliotropium antillanum Urb., Symb. Antill. 4: 528. 1910. Euploca baclei (DC.) Diane & Hilger comb. nov. º Heliotropium baclei DC., Prodr. 9: 546. 1845. Euploca ballii (Domin) Diane & Hilger comb. nov. º Heliotropium ballii Domin, Biblioth. Bot. 89: 1098. 1928. Euploca bursifera (C.Wright) Diane & Hilger comb. nov. º Heliotropium bursiferum C.Wright, in Griseb., Cat. Pl. Cub.: 211. 1866. 48 H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae Euploca campestris (Griseb.) Diane & Hilger comb. nov. º Heliotropium campestre Griseb., Abh. Königl. Ges. Wiss. Göttingen 19: 186. 1874. For further synonyms see Förther (1998). Euploca chrysantha (Phil.) Diane & Hilger comb. nov. º Heliotropium chrysanthum Phil., Anales Univ. Chile 21: 401. 1862. Euploca cupressina (Craven) Diane & Hilger comb. nov. º Heliotropium cupressinum Craven, Austral. Syst. Bot. 9: 570: 1996. Euploca humifusa (Kunth) Diane & Hilger comb. nov. º Heliotropium humifusum Kunth, in Humborldt, Bonpland & Kunth, Nov. Gen. Sp. 3: 85. 1818. For further synonyms see FÖRTHER (1998). Euploca hypogaea (Urb. & Ekman) Diane & Hilger comb. nov. º Heliotropium hypogaeum Urb. & Ekman, Ark. Bot. 22A, no.10: 105. 1929. º Hilgeria hypogaea (Urb. & Ekman) Förther, Sendtnera 5: 133. 1998. Euploca lagoënsis (Warm.) Diane & Hilger comb. nov. º Heliotropium lagoënse (Warm.) Gürke, in Engler & Prantl, Nat. Pflanzenfam. 4(3a): 97. 1893. º Schleidenia lagoënsis Warm., Vidensk. Meddel. Dansk Naturhist. Foren. Kjobenhavn 1867: 15. 1867. For further synonyms see FÖRTHER (1998). Euploca mendocina (Phil.) Diane & Hilger comb. nov. º Heliotropium mendocinum Phil., Anales Univ. Chile 21: 400. 1862. Euploca ovalifolia (Forssk.) Diane & Hilger comb. nov. º Heliotropium ovalifolium Forssk., Fl. Aegypt.-Arab.: 38. 1775. Euploca procumbens (Mill.) Diane & Hilger comb. nov. º Heliotropium procumbens Mill., Gard. Dict., ed. 8: no. 10. 1768. For further synonyms see FÖRTHER (1998). Euploca pulvina (Craven) Diane & Hilger comb. nov. º Heliotropium pulvinum Craven, Austral. Syst. Bot. 9: 577. 1996. Euploca rariflora (Stocks) Diane & Hilger comb. nov. º Heliotropium rariflorum Stocks, J. Bot. (Hooker) 4: 174. 1852. Euploca rariflora subsp. hereroensis (Schinz) Diane & Hilger comb. nov. º Heliotropium hereroense Schinz, Vierteljahresschr. Naturf. Ges. Zürich 60: 404. 1915. º Heliotropium rariflorum Stocks subsp. hereroense (Schinz) Verdc., Fl. Trop. East Afr., Boraginaceae: 70. 1991 For further synonyms see FÖRTHER (1998). H.H. Hilger & N. Diane, Systematic analysis of Heliotropiaceae 49 Euploca serpylloides (Griseb.) Diane & Hilger comb. nov. º Heliotropium serpylloides Griseb., Cat. Pl. Cub.: 212. 1866. º Hilgeria serpylloides (Griseb.) Förther, Sendtnera 5: 133. 1998. Euploca strigosa (Willd.) Diane & Hilger comb. nov. º Heliotropium strigosum Willd., Sp. Pl. ed.4 1(2): 743. 1798. For further synonyms see FÖRTHER (1998). Euploca styotricha (Craven) Diane & Hilger comb. nov. º Heliotropium styotrichum Craven, Austral. Syst. Bot. 9: 580. 1996. Euploca tenuifolia (R.Br.) Diane & Hilger comb. nov. º Heliotropium tenuifolium R.Br., Prodr.: 494. 1810. For further synonyms see FÖRTHER (1998). Acknowledgement The authors wish to thank the following collectors who provided us with plant material: LYN CRAVEN (Canberra, Australia), TASSILO FEUERER (Hamburg), HARALD FÖRTHER (München), MARIA HOFMANN (Berlin), MATHIAS JENNY (Frankfurt), ANNA KAGIAMPAKI (Iraklion, Greece), NORBERT KILIAN (Berlin), SIGRID LIEDE (Bayreuth), MONIKA LÖSCHNER (Berlin), JAMES MILLER (St. Louis MO, USA), WOLFGANG SCHULTKA (Gießen), HAGEN STENZEL (Berlin), HANS-JÜRGEN TILLICH (München), MAXIMILIAN WEIGEND (Berlin), ELKE ZIPPEL (Berlin).We thank the Botanische Staatssammlung München (M), the Botanischer Garten and Botanisches Museum Berlin-Dahlem (B), the Herbarium Senckenbergianum, Frankfurt (FR), the Herbarium University Kassel (KAS), the Royal Botanic Gardens, Kew (Great Britain), the Australian National Herbarium, Canberra (CAN), and the Missouri Botanical Garden Herbarium (MO) for plant material. 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VAN DE PEER, Y. & DE WACHTER, R. 1994: TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. — Comput. Appl. Biosci. 10: 569–570. VERDCOURT , B. 1991: Boraginaceae. — In: POLHILL, R.M. (ed.), Flora of tropical East Africa. 1–124. — Rotterdam and Brookfield. VERDCOURT , B. 1987: A new genus Nogalia (Boraginaceae-Heliotropioideae) from Somaliland and southern Arabia. — Kew Bull. 43: 431–435. Accepted for publication July 16, 2003 Addresses of the authors: HARTMUT H. HILGER and NADJA DIANE, Institut für Biologie — Systematische Botanik und Pflanzengeographie, Freie Universität Berlin, Altensteinstr. 6, D-14195 Berlin, Germany; e-mail; diane@zedat.fu-berlin.de; hahilger@zedat.fu-berlin.de