Phylogeny and Evolution of Basils and Allies (Ocimeae, Labiatae) Based On Three Plastid DNA Regions

MOLECULAR PHYLOGENETICS AND EVOLUTION
Molecular Phylogenetics and Evolution 31 (2004) 277299 www.elsevier.com/locate/ympev
Phylogeny and evolution of basils and allies (Ocimeae, Labiatae) based on three plastid DNA regions
Alan J. Paton,a,* David Springate,b Somran Suddee,a,1 Donald Otieno,c Ren ee J. Grayer,b a a b Madeline M. Harley, Fiona Willis, Monique S.J. Simmonds, Martyn P. Powell,b and Vincent Savolainenb
c
The Herbarium, Royal Botanic Gardens, Kew, Richmond TW9 3AB, UK Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond TW9 3AB, UK Charles E. Moss Herbarium, School of Animal, Plant, and Environmental Science, University of the Witwatersrand, Private Bag 3, Wits 2050, Gauteng Province, South Africa
b
Received 8 April 2003; revised 1 August 2003
Abstract A phylogeny of basils and allies (Lamiaceae, tribe Ocimeae) based on sequences of the trnL intron, trnLtrnF intergene spacer and rps 16 intron of the plastid genome is presented. Several methods were used to reconstruct phylogenies and to assess statistical support for clades: maximum parsimony with equally and successively weighted characters, bootstrap resampling, and Bayesian inference. The phylogeny is used to investigate the distribution of morphological, pericarp anatomy, chemical, and pollen characters as well as the geographical distribution of the clades. Tribe Ocimeae is monophyletic and easily diagnosable with morphological synapomorphies. There are monophyletic clades within Ocimeae that broadly correspond to currently recognised subtribes: Lavandulinae, Hyptidinae, Ociminae, and Plectranthinae. Only Lavandulinae has clear non-molecular synapomorphies. Several currently recognised genera are not monophyletic. Floral morphology consistent with sternotribic pollination is most common in Ocimeae, but there are independent departures from this model. Buzz pollination is likely in some species, the only postulated occurrence of this within Lamiaceae. Quinone diterpenoids and avones in the leaf exudates dier in their distributions across the phylogeny and this could contribute to dierences in the recorded medicinal as well as pesticidal uses of the species in the dierent clades. Mapping geographic distribution on to an ultrametric phylogenetic tree produced using non-parametric rate smoothing supports an Asiatic origin for Ocimeae. There are several secondary occurrences in Asia arising from the African Ociminae and Plectranthinae clades. Colonisation of Madagascar occurred at least ve times, and New World colonisation occurred at least three times. 2003 Elsevier Inc. All rights reserved.
1. Introduction Basils and their allies, classied as tribe Ocimeae Dumort. in Lamiaceae, subfamily Nepetoideae, are a predominantly tropical group containing 35 genera and 1060 species. There are main centres of diversity in Tropical Africa and Madagascar, China and Indochina, and in South America (Cantino et al., 1992; Harley
* Corresponding author. Fax: +44-181-322-5197. E-mail address: a.paton@rbgkew.org.uk (A.J. Paton). 1 Present address: Forest Herbarium, Royal Forest Department, Bangkok 10900, Thailand.
et al., in press). The group is economically and medicinally important. Basil (Ocimum) and some species of Plectranthus are widely used as pot herbs. Commercially around 100 tonnes of essential oil are produced annually from species of Ocimum and over 400 tonnes from Lavender (Lavandula) (Lawrence, 1992; Morhy et al., 1970). Medicinal uses include pain relief and anti-cancer ~ez properties (Githinji and Kokwaro, 1993; Riviera-Nun n De Castro, 1992). and Obo Several genera currently recognised within Ocimeae, such as Plectranthus, have no clear synapomorphies and could be polyphyletic. Although a few molecular phylogenic trees of Lamiaceae and subfamily Nepetoideae have been produced using plastid DNA restriction site
1055-7903/$ - see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2003.08.002
278
A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299
data (Wagsta et al., 1995), rbcL (Kaufmann and Wink, 1994), rbcL and ndhF (Wagsta et al., 1998), none deal in any detail with Ocimeae. Even in the cases of Ocimum and Platostoma where morphological parsimony analyses have been published (Paton, 1997; Paton et al., 1999), nodes lack statistical support. Robust phylogenetic analyses are much needed in order to eciently explore the medicinal and economic uses of the group. The aims of this paper are rst, to provide a phylogenetic tree of tribe Ocimeae based on three plastid DNA regions (trnL intron, trnLtrnF intergene spacer, and rps16) and to evaluate the monophyly of the tribe; second, to use the phylogeny to investigate the distribution of morphological, chemical, and pollen characters as well as the geographical distribution of the clades.
2. Materials and methods 2.1. Sampling and data gathering A representative sample of tribe Ocimeae was selected using 122 species (12% of all species) from 27 genera (80% generic representation). To evaluate the monophyly of Ocimeae, the analysis also included a representative sample of 12 species from the remainder of subfamily Nepetoiodeae, covering the subtribes Nepeteae, Mentheae, and Elsholtzieae (Cantino et al., 1992; Harley et al., in press). Three taxa from subfamily Prostantheroideae, one from Symphorematoideae, and six from Viticoideae were selected as outgroups for all analyses following the phylogenetic analysis from Wagsta et al. (1998). The list of plant samples with voucher information and GenBank/EMBL Accession numbers is provided in Table 1. Total DNA was extracted from fresh, silica gel-dried, and herbarium specimens using the 2 CTAB method of Doyle and Doyle (1987) and subsquently puried through caesium chloride gradient. The trnLtrnF region (trnL intron and trnLtrnF intergene) was amplied with primers c (50 -CGAAATCGGTAGACGCTACG-30 ) and f (50 -ATTTGAACTGGTGACACGAG-30 ) from Taberlet et al. (1991) using the following PCR program: 30 cycles, 1 min denaturation at 95 C, 30 s annealing at 50 C, 1 min extension at 72 C, and 7 min nal extension. The rps16 intron was amplied using primers rpsF (50 -GT GGTAGAAAGCAACGTGCGACTT-30 ) and rps2R (50 -TCGGGATCGAACATCAATTGCAAC-30 ; Oxelman et al., 1997). Amplication products were then puried using QIAquick columns (Qiagen). DNA cycle sequencing with BigDye terminators (PE Applied Biosystems) and PCR primers was performed in 5 ll volumes on the cleaned PCR products (26 cycles, 10 s denaturation at 96 C, 5 s annealing at 50 C, and 4 min extension at 60 C). Cycle sequencing reactions were puried by ethanol precipitation and run on an Applied Biosystems 377
automated DNA sequencer following the manufacturers protocols (PE Applied Biosystems); both strands were sequenced and provided >75% overlap. Sequences were aligned by eye and indels coded using PAUP gap (Cox, 1997); nested gaps were coded as separate events. Morphological data were gathered from herbarium specimens and living collections. Thirty-three macromorphological characters were identied (Table 2). Pericarp characters within Ocimeae are well documented (Ryding, 1992, 1993, 1995). Five pericarp characters were employed in the analysis and included in the morphological dataset (Table 2, characters 3438). Taxa were scored according to the observations cited in the above references. The morphological matrix is appended (Appendix A). Pollen morphology from all genera studied was examined following the methods described in Harley (1992). Selecting non-ambiguous pollen characters for cladistic analysis of Ocimeae is problematical as many characters are continuously variable. Pollen morphology will be discussed in detail in another paper, but features that may represent synapomorphies of particular clades are indicated in the discussion. 2.2. Molecular and morphological phylogenetic analyses We used several methods to reconstruct phylogenies and to assess statistical support for clades: maximum parsimony with equally and successively weighted characters, bootstrap resampling, and Bayesian inference. Maximum parsimony analysis was performed on the trnLF region and rps 16 intron combined and separately and on the morphological data separately using the program PAUP*4.0b10 (Swoord, 2001). Mostparsimonious trees with molecular and morphological characters were obtained from 1000 replicates of random taxon addition using equally weighted (EW) characters (Fitch, 1971) and tree bisectionreconnection (TBR) branch swapping with 10 trees held at each step. These trees were also used to reweight the characters according to the best t of their rescaled consistency indices (Farris, 1989), and new searches as described above were performed using the successive weights (SW) until equilibrium between number of trees and weights was reached; this approach in tree search reduces the disturbing eect, if any, of unstable taxa (Farris, 1969). Morphological data were also optimised on one of the EW trees produced from the analysis of all DNA regions combined in McClade 3.08 (Maddison and Maddison, 1999) with ACCTRAN optimisation and polytomies treated as hard polytomies. Individual characters were traced on the tree to identify morphological synapomorphies. Bootstrap (BS) resampling (Felsenstein, 1985) was performed on the trnLF region and rps16 intron
A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299 Table 1 List of plant samples with voucher information and GenBank/EMBL Accession numbers trnLtrnF Genbank Accession Aeollanthus buchnerianus Briq. Aeollanthus densiorus Ryding Alvesia rosmarinifolia Welw. Anisochilus harmandii Doan Anisochilus pallidus Benth. Basilicum polystachyon (L.) Moench Callicarpa americana L. Callicarpa japonica Thunb. Capitanopsis angustifolia (Moldenke) Capuron Catoferia chiapsis Benth. Clinopodium vulgare L. subsp. arundanum Congea tomentosa Roxb. Dauphinea brevilabra Hedge Elsholtzia stauntonii Benth. Endostemon obtusifolius (Benth.) N.E.Br. Fuerstia africana T.C.E.Fr. Gmelina hystrix Kurz Haumaniastrum katangense (S.Moore) Duvign. & Plancke Hemizygia albiora (N.E.Br.) Ashby Hemizygia foliosa S.Moore Hemizygia incana Codd Hemizygia modesta Codd Hemizygia obermeyerae Ashby Hemizygia parvifolia Codd Hemizygia persimilis (N.E.Br.) Ashby rke) Ashby Hemizygia pretoriae (Gu Hemizygia punctata Codd Hemizygia stalmansii A.J.Paton & K.Balkwill rke) Ashby Hemizygia subvelutina (Gu Hemizygia transvaalensis (Schltr.) Ashby Hemizygia teucriifolia (Hochst.) Briq. Lavandula buchii Webb & Berthel. Lavandula maroccana Murb. Lavandula minutolii C. Bolle Lavandula rotundifolia Benth. Melissa ocinalis L. Mentha suaveolens Ledeb. Nepeta ssa C.A.Mey. Nepeta menthoides Boiss. & Buhse Nepeta racemosa Lam. Nepeta straussii Hausskn. & Bornm. Ocimum americanum L. var. pilosum (Willd.) A.J.Paton Ocimum basilicum L. Ocimum citriodorum Vis. Ocimum lamentosum Forssk. Ocimum gratissimum L. var. gratissimum Ocimum gratissimum L. var. macrophyllum Briq. 1 Ocimum gratissimum L. var.macrophyllum Briq. 2 Ocimum labiatum (N.E.Br.) A.J.Paton1 Ocimum labiatum (N.E.Br.) A.J.Paton2 Ocimum selloi Benth. Ocimum serratum (Schltr.) A.J.Paton Ocimum tenuiorum L. Origanum vulgare L. Orthosiphon aristatus (Blume) Miq. Orthosiphon parishii Prain Orthosiphon rotundifolius Doan Orthosiphon rubicundus (D.Don) Benth. Platostoma africanum P.Beauv. AJ505434 AJ505435 AJ505436 AJ505437 AJ505438 AJ505439 AJ505535 AJ505536 AJ505440 AJ505537 AJ505547 AJ505530 AJ505441 AJ505526 AJ505442 AJ505550 AJ505527 AJ505540 AJ563764 AJ563765 AJ563766 AJ563767 AJ563768 AJ563769 AJ563770 AJ563771 AJ563772 AJ563773 AJ563774 AJ563775 AJ563776 AJ505460 AJ505461 AJ505462 AJ505463 AJ505529 AJ505541 AJ505430 AJ505431 AJ505432 AJ505433 AJ505464 AJ505465 AJ505548 AJ505466 AJ505467 AJ505468 AJ505469 AJ505470 AJ505471 AJ505542 AJ505472 AJ505473 AJ505543 AJ505474 AJ505475 AJ505476 AJ505477 AJ505478 rps16 Genbank Accession AJ505327 AJ505328 AJ505329 AJ505330 AJ505331 AJ505332
279
Voucher Cult., K-1970-2734, Brummitt 10401, (K) Cult., K-1970-3760, Mathew 6137 (K) Harder, Luwilka & Zimba 3634 (K) Suddee 775 (BKF, K, TCD) Suddee et al.1080 (BKF, K, TCD) Greenway & Kanuri 14501 (K) Cult., K-0818400507 (K) Cult., K-1934-12904 (K) Clement, Phillipson & Mantanantsoa 2117 (K) Cult., K-1983-661, Chase 1149 (K) Cult., K-453-79-04649 (K) Wagsta, s.n. (BHO) (Wagsta and Olmstead, 1996) Cult., K-1998-2417, Hardy & Rauh 2876 (K) Wagsta 356, (BHO) (Wagsta et al. 1995) Cult., K-1977-4733, Goyder & Paton 4105 (K) G. Simon & M. Meliyo 228 (K) Cult., K-381-74-02999 (K) Cult., K-1995-1197, Chase 13330 (K) Otieno 66 (J) Otieno 45 (J) Otieno 75 (J) Otieno 120 (J) Otieno 13 (J) Otieno 71 (J) Otieno 76 (J) Otieno 50 (J) Otieno 72 (J) Otieno 111 (J) Otieno 69 (J) Otieno 56 (J) Otieno 64 (J) Upson 299 (RNG) Upson, s.n. (RNG) Upson s.n (RNG) Upson s.n (RNG) Wagsta 88-09 (BHO) (Wagsta et al., 1995) Cult., K-1970-3169 (K) Jamzad & Nikchehreh 80486 (TARI) Jamzad s.n. (K) Jamzad s.n. (TARI) Jamzad et al. 76846 (TARI) Cult., Suddee 1145 (K) Cult., Suddee et al. 894 (BKF,K) Chase 9939 (K) Brummitt 18993 (K) Cult., Suddee & Meade 1139 (BKF) Cult., K-1993-1030, Schellingerhaut s.n. (K) Cult., Suddee et al. 891/1 (BKF, K) Edwards s.n. (K, NU) Balkwill et al. 10848 (K) Gentry & Zardinini 49819 (K) Balkwill et al. 10861 (J, K) Cult., Suddee et al.893 (BKF, K, TCD) Cult., K-000-69-19317, Chase 13334 (K) Suddee et al. 969 (BKF, K, TCD) Suddee & Puudjaa 1118 (BKF, K, TCD) Suddee et al. 944 (BKF, K, TCD) Suddee 809 (BKF, TCD) Pawek 4584 (K)
AJ505412 AJ505333 AJ505414 AJ505426 AJ505411 AJ505334 AJ505406
AJ505427 AJ505407 AJ505417 AJ563783 AJ563784 AJ563785 AJ563786 AJ563787 AJ563788 AJ563789 AJ563790 AJ563791 AJ563792 AJ563793 AJ563794 AJ563795 AJ505346 AJ505347 AJ505348 AJ505349 AJ505410 AJ505418 AJ505323 AJ505324 AJ505325 AJ505326 AJ505350 AJ505351 AJ505420 AJ505352 AJ505353 AJ505354 AJ505355
AJ505356 AJ505419 AJ505357 AJ505358 AJ505422
AJ505359
AJ505360
280 Table 1 (continued)
trnLtrnF Genbank Accession Platostoma annamense (G.Tayl.)A.J.Paton Platostoma calcaratum (Hemsl.) A.J.Paton var. garretii (Craib) S.Suddee, in ed. Platostoma cambodgense (Doan) A.J.Paton var. cambodgense Platostoma cambodgense (Doan) A.J.Paton var.subulatum S.Suddee in ed. Platostoma cochinchinense (Lour.) A.J.Paton Platostoma coeruleum (R.E.Fr.) A.J.Paton Platostoma coloratum (D.Don) A.J.Paton var. coloratum Platostoma coloratum (D.Don) A.J.Paton var. minutum S.Suddee in ed. Platostoma mbriatum A.J.Paton Platostoma hildebrandtii (Vatke) A.J.Paton Platostoma hispidum (L.) A.J.Paton Platostoma intermedium A.J.Paton Platostoma kerrii (Doan) A.J.Paton Platostoma mekongense S.Suddee in ed. Platostoma ocimoides (G.Tayl.) A.J.Paton Platostoma rotundifolium (Briq.) A.J.Paton Platostoma rubrum (Doan) A.J.Paton Platostoma siamense (Murata) A.J.Paton Platostoma tectum A.J.Paton Plectranthus albicalyx S.Suddee in ed. rke Plectranthus alboviolaceus Gu Plectranthus ambonicus (Lour.) Spreng. Plectranthus barbatus Andr. Plectranthus buchananii Bak. Plectranthus calycinus Benth. Plectranthus ciliatus E.Mey rke) Agnew Plectranthus coeruleus (Gu Plectranthus crassus N.E.Br. Plectranthus cylindraceus Benth. Plectranthus fredricii (G.Taylor) A.J.Paton, in ed. Plectranthus glabratus (Benth.) Alston Plectranthus helferi Hook. f. 1 Plectranthus helferi Hook. f. 2 Plectranthus laxiorus Benth. Plectranthus oertendahlii T.C.E. Fr. Plectranthus parishii Prain Plectranthus petiolaris Benth. Plectranthus puberulentus J.K.Morton 1 Plectranthus puberulentus J.K.Morton 2 Plectranthus sanguineus Britten Plectranthus scutellaroides (L.) R.Br. Plectranthus thyrsoideus (Bak.) B.Matthew Plectranthus xerophilus Codd Prosthanthera nivea Benth. Prosthanthera petrophila B.J.Conn Puntia stenocaulis Hedge Pycnostachys reticulata (E.Mey.) Benth. Pycnostachys umbrosa (Vatke) Perkin Pycnostachys urticifolia Hook. Rosmarinus ocinalis L. Salvia guaranitica Benth Syncolostemon argenteus N.E.Br Syncolostemon comptonii Codd Syncolostemon abellifolius (S.Moore) A.J.Paton rke) Ashby Syncolostemon macranthus (Gu Syncolostemon parviorus Benth. AJ505479 AJ505480 AJ505481 AJ505482 AJ505483 AJ505484 AJ505485 AJ505486 AJ505487 AJ505488 AJ505489 AJ505490 AJ505491 AJ505492 AJ505493 AJ505494 AJ505495 AJ505496 AJ505497 AJ505498 AJ505531 AJ505499 AJ505500 AJ505501 AJ505502 AJ505532 AJ505503 AJ505504 AJ505538 AJ505505 AJ505508 AJ505509 AJ505552 AJ505510 AJ505534 AJ505511 AJ505512 AJ505506 AJ505507 AJ505513 AJ505514 AJ505533 AJ505515 AJ505524 AJ505525 AJ505545 AJ505516 AJ505517 AJ505518 AJ505546 AJ505549 AJ563777 AJ563778 AJ563779 AJ563780 AJ563781
rps16 Genbank Accession AJ505361
Voucher Suddee et al.1028 (BKF) Suddee et al.910 (BKF, K, TCD) Chalermklin & Meade 98-10-16-01 (BKF) Suddee 767 (BKF, K, TCD) Wongprasert, Suddee & Puudjaa s.n (BKF, K, TCD) Bidgood et al.3835 (K) Suddee et al.958 (BKF, K, TCD) Suddee et al.915 (BKF, K, TCD) Suddee 823 (BKF, K, TCD) Bally & Smith B14742 (K) Beusekom et al. 3624 (K) Suddee et al.947 (BKF, K, TCD) Suddee 769 (BKF, K, TCD) Chalermklin & Meade 98-10-16-02 (BKF, K) Suddee 788 (BKF, K, TCD) Gilbert & Phillips 9239 (K) Suddee et al.998 (BKF, K, TCD) Suddee & Pooma 849 (BKF, K, TCD) Suddee at al 1005 (BKF, K, TCD) Suddee et al.868 (BKF, K, TCD) Cult., K-1970-3926, Chase 9315 (K) Suddee at al 869 (BKF) Cult., K1982-5914, Thulin 4380 (K) Cult., K-1970-3559, Brummitt 11597 (K) Balkwill et al.10880 (J, K) Cult., K-1991-6, Chase 13336 (K) Cult., K-1955-42604, Delap s.n. (K) Cult., K-1970-2059, Brummitt 9700 (K) Cult., K-1996-1453, Chase 8518 (K) Cult., K-1999-15, RHS Wisley (K) Wongprasert, Suddee & Puudjaa s.n (BKF, K, TCD) Suddee & Puudjaa 1098 (BKF, K, TCD) Chase 9768 (K) Edwards s.n (K) Cult., K-1969-5789, Chase 3380 (K) Suddee 11444 (BKF, K, TCD) Cult., K-1996-2729, U of Natal (K) K-1970-3763, Mathew 6158 (K) Cult., K-1970-3784, Mathew 6830 (K) Cult., K-1970-2072, Brummitt s.n (K) Cult., Suddee et al.1094 (BKF, K, TCD) Cult., K-5638704012, Chase 13332 (K) Cult., k-1989-1322, Hardy 6735 (K) M.W. Chase 6980 (K) M.W.Chase 6975 (K) M T Thulin 10506 (UPS) Cult., K-1999-2425, Nat Bot. Gar. S. Africa (K) Cult., k-1970-3755, Mathew 6067 (K) Cult., K-1999-2426, Nat. bot. gar. s.Africa (K) Cult., K-1975-1177, Chase 13331 (K) Cult., K-1973-14217 (K) Otieno 93 (J) McCallum 916 (J) Linder 3981 (J) Otieno 109 (J) Otieno 84 (J)
AJ505362 AJ505363 AJ505364 AJ505365 AJ505366 AJ505367 AJ505368

AJ505369 AJ505370 AJ505371
AJ505372 AJ505373 AJ505374 AJ505375 AJ505376 AJ505377 AJ505377 AJ505378 AJ505379 AJ505380 AJ505409 AJ505381 AJ505382 AJ505383 AJ505384 AJ505387 AJ505388 AJ505429 AJ505389
AJ505390 AJ505391 AJ505385 AJ505386 AJ505392 AJ505393 AJ505405 AJ505394 AJ505403 AJ505404 AJ505424 AJ505395 AJ505396 AJ505397 AJ505425 AJ505421 AJ563796 AJ563797 AJ563798 AJ563801 AJ563800
A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299 Table 1 (continued) trnLtrnF Genbank Accession Syncolstemon rotundifolius Benth. Tectona grandis L. f. Tetradenia nervosa Codd Tetradenia fruticosa Benth. Thorncroftia longifolia N.E.Br. Thorncroftia media Codd Thymus serpyllum L. var. citriodorum Vitex trifolia L. AJ505523 AJ505528 AJ505520 AJ505519 AJ505521 AJ505522 AJ505544 AJ505539 rps16 Genbank Accession AJ505402 AJ505408 AJ505399 AJ505398 AJ505401 AJ505400
281
Voucher Balkwill & Manning 414 (K) Waimea 73P172 (Wagsta and Olmstead, 1996) Cult., K-1993-3116, Hardy 2910B (K) Cult., K-1989-1324, Hardy 2910A (K) McDade LM 1281 (J) Cult., K-1993-3115, Hardy 3966 (K) Cult., K-1975-1177, Chase 13331 (K) TCMK 15, Chase 8757 (K)
AJ505416
Table 2 Characters and coding used in the morphological MP analysis and mapping morphological and chemical characters and geographical distribution to the phylogeny CI 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Fertile bracts: uniformly green (0); basally coloured (1) Peduncle: absent (0); present (1) Cyme branching: diachasial (0); cincinnate (1); inapplicable (2) No. of owers in cyme: 1 (0); 13 variable (1); 3 uniform (2); >3 (3) Bracteoles: present (0); absent (1) Cyme positioning: opposite (0); spiral (1); one-sided (2) Cyme attachment: pedicellate (0); absent (1); sessile by pedicel fusion to calyx (2) Pedicel attachment: median (0); opposite posterior lip (1) Calyx circumscissile: not circumscissile (0); circumscissile (1) Position of lateral calyx lobe: median, between posterior and anterior (0); close to posterior, not in same plane (1); close to posterior, in same plane (2); close to anterior (3); absent (4) Division of anterior calyx lobes: to same depth as lateral and anterior (0); fusedto less depth than anterior/lateral split (1); splitto a greater depth than anterior/lateral split (2). Shape of anterior calyx lobes: lanceolate (0); subulate (1); spinescent (2); sinuate (3); rounded (4); inconspicuous (5) Base of corolla tube: parallel sided (0); gibbous (1); saccate (2); spurred (3) Lateral corolla lobe position: forming part of posterior lip (0); freelateral (1); part of anterior lip (2); absent (3) Posterior/anterior corolla lip ratio: > or equal to 0.8 (0); <0.8 (1) Tube shape: straight (0); curved down (1); sigmoid (2) Anterior lobe hinged: absent (0); present (1) Anterior lobe reexed after anthesis: absent (0); present (1) Stamen position: ascending (0); declinate (1); spreading porect (2) Length of stamen: posterior exceeding anterior (0); anterior exceeding posterior (1); equal (2) Fusion of stamens: no staminal fusion (0); all stamens fused, with the two anterior stamens fused together and to the adjacent posterior stamen (1); anterior stamens not fused together but each anterior stamen attached to its adjacent posterior stamen (2) Anterior stamen fusion: absent, no fusion (0); present, fusion of the anterior pair of stamens only, this pair separated from the posterior stamens (1) Attachment of posterior stamen: Base to mid (0); mid to throat (1); throatanterior side (2); throat regular separation (3) Posterior lament form: taperingno appendage (0); small tuft of hairs at base (1); basally swollen (2); appendiculatearticulate (3); appendiculateattened (4); bent not appendiculate (5) Anthers: with convergent thecae (0); synthecous (1); parallel (2) Style: bid (0); clavate (1); capitatebroad lobes (2); emarginate (3); bid unequal (4) Stylopodium: absent (0); present (1) Style shield: absent (0); present (1) Disk lobing: equally lobed (0); anterior larger (1), lobes opposite nutlets, not applicable (2) Position of disk lobes: alternate with nutlets (0); opposite nutlets (1) Nutlet areole: present (0); absent (1); not applicable, carpel not divided (2) Fruit: divided into mericarps (0); undivided (1). Anthers attachment: Thecae lateral (0); dorsixed (1). 0.33 0.15 0.12 0.25 0.25 1.0 0.33 0.5 0.5 0.28 0.13 0.26 0.2 0.18 0.09 0.14 1.0 0.04 0.4 0.33 0.4 RI 0.89 0.65 0.7 0.55 0.91 1.0 0.69 0.95 0.5 0.7 0.66 0.74 0.6 0.76 0.74 0.74 1.0 0.52 0.76 0.52 0.83 RC 0.3 0.1 0.08 0.16 0.23 1.0 0.23 0.48 0.25 0.20 0.09 0.19 0.12 0.14 0.07 0.11 1.0 0.02 0.34 0.17 0.33
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
1.0 0.38 0.38 0.67 0.27 0.5 0.33 0.13 1.0 0.4 0.5 1.0 1.0 1.0 0.56
1.0 0.86 0.81 0.94 0.53 0.5 0.0 0.84 1.0 0.87 0.8 1.0 1.0 1.0 0.91
1.0 0.32 0.3 0.63 0.14 0.25 0.0 0.11 1.0 0.35 0.4 1.0 1.0 1.0 0.51
Pericarp anatomy characters 34. Number of cells in schlerenchyma region: >1 (0); 1 (1) 35. Shape of cells in schlerenchyma region: star- or stone-shaped (0); bone-shaped arranged vertically (1) 36. Crystals in schlerenchyma region: absent (0); present (1)
282 Table 2 (continued)
CI 37. Plate-shaped content in exocarp cells: absent (0); present (1) 38. Postion of lumen in the bone cells: basal to central (0); apical (1); inapplicable (2) Chemical characters mapped to phylogeny 39. 5-OH-6,7-diOMe-Flavones absent (0); present (1) 40. 5-OH-6,7,8-triOMe-Flavones absent (0); present (1) 41. 5,6-diOH-7,8-diOMe-Flavones absent (0); present (1) 42. Flavonols absent (0); present (1) 43. Quinonoid diterpenoids absent (0); present (1) 44. Geographic distribution Tropical Africa (0); Madagascar (1); SE Asia (2); Mediterranean/Central Asia (3); Macaronesia (4); Australia (5); New World (6) 0.25 0.4 0.33 0.5 1.0 0.25 0.5 0.42
RI 0.75 0.81 0.75 0.92 1.0 0.0 0.89 0.76
RC 0.19 0.33 0.25 0.46 1.0 0.0 0.44 0.33
Statistics were calculated based on the characters t to the topology of the equally weighted molecular tree shown in Fig. 3.
combined and on morphological characters separately, using PAUP* 4.0b10 (Swoord, 2001). We performed in each case 500 replicates with TBR swapping algorithm, with EW and SW, and holding ve trees per replicate, under maximum parsimony (MP) optimality criterion. Bayesian phylogeny reconstruction was performed using the program MrBayes 2.01 and available at http:// www.morphbank.ebc.uu.se/mrbayes/ (Huelsenbeck and Ronquist, 2001). For the two DNA regions combined, we performed 500,000 generations on four Markov chains, trees sampled every hundred generations and uniform prior distribution (mcmc ngen 500,000, printfreq 100, samplefreq 10, nchains 4). Because HKY85 (Hasegawa et al., 1985) is a good compromise between the complexity of the model of DNA substitution (transition/transversion ratio plus dierential nucleotide composition) and time requirement for its computation, we set up this model as follows: lset nst 2 rates equal. The rst 15,000 trees were discarded to allow for the burn-in of the process. Biogeographic relationships were explored by optimising the geographic distribution of terminal taxa (Table 2, Appendix A) on a ultrametric tree in McClade 3.08 (Maddison and Maddison, 1999) with ACCTRAN optimisation and polytomies treated as hard polytomies. The ultrametric tree was produced using the non-parametric rate smoothing (NPRS) method of Sanderson (1997) implemented in TreeEdit (v1.0 alpha 4-61, written by A. Rambaut and M. Charleston; http://www. evolve.zps.ox.ac.uk/software/TreeEdit/main.html). Maximum parsimony EW branch lengths resulting from the molecular analysis were optimised onto the trees before NPRS. 2.3. Chemical characters Flavonoid and diterpene constituents were studied in 19 species of Plectranthus and related genera (Pycnostachys, Tetradenia, Thorncroftia, Aeollanthus, and Dauphinea), one species each of Endostemon and Basilicum. Results are also included from ve species of
Ocimum (Grayer et al., 1996; Grayer et al., 2001), four species of Lavandula (Marin et al., unpublished results), four species of Nepeta (Jamzad et al., 2003), one species of Thymus (Marin et al., 2003), and one each of Clinopodium, Mentha, and Origanum (Marin et al., unpublished). Fresh leaves (ca. 2 g) of each species were extracted with diethyl ether and studied by HPLC with diode array detection and atmospheric pressure chemical ionisation mass spectrometry as described previously (Grayer et al., 2001). HPLC chromatograms were examined at dierent wavelengths (270, 335, and 420 nm) suitable to detect both exudate avonoids and quinonoid diterpenoids, and the presence or absence of these compounds was scored. Exudate avonoids were grouped on the basis of their UV spectra into avones and avonols (Mabry et al., 1970) and identied according to the HPLC method developed by Grayer et al. (2001) and by cochromatography with standards. The avones were further subdivided according to the three main A-ring hydroxylation/methoxylation substitution patterns found in these species, 5-hydroxy-6, 7-dimethoxyavones (5-OH-6,7-diOMe), 5-hydroxy-6,7, 8-trimethoxyavones (5-OH-6,7,8-triOMe), and 5,6-dihydroxy-7,8-dimethoxyavones (5,6-diOH-7,8-diOMe). To score the presence or absence of quinonoid diterpenoids, the colour of the extract was observed (only orange extracts contain appreciable amounts of these constituents) and the HPLC chromatograms were studied to see whether they contained peaks with the UV/Vis spectra characteristic of these diterpenoids (maxima in both UV and visible light). They were not identied further, nor subdivided into dierent classes. The distribution of avonoids and quinonoid diterpenoids are provided in Appendix A. Data were optimised on the molecular phylogeny following the same method as for the morphological characters (see Table 2). The characters were scored as missing for taxa not studied.
283
3. Results 3.1. MP analyses After performing phylogenetic analyses of the trnL trnF and rps16 regions separately, no hard incongruences were found (no contradictory clades supported by bootstrap >60%; data not shown). The congruence between the two data sets supports the likelihood that they track the same phylogeny (Elden as and Linder, 2000) and therefore sequence data from the two regions were analysed together. The results of the MP analysis of DNA partitions combined with gaps coded and with the characters successively weighted are shown in Figs. 1AC. Only bootstrap support (BS) greater than 50%, calculated with EW are reported. The EW analysis produced 40 trees with CI 0.56 and RI 0.76. The SW analysis produced 656 trees with CI 0.92 and RI 0.95. The major dierence between the EW and SW analyses is that there is much less resolution in the Ociminae clade in the EW analysis. However, the resolution in this clade provided by the SW analysis lacks BS > 50% (Fig. 1B).
The monophyly of the Ocimeae clade is supported (BS 99%, Fig. 1A). The position of Elsholtzia as sister group to tribe Ocimeae is also supported (BS 62%). Lavandula is sister to the remainder of the Ocimeae clade with both clades well supported (Lavandula BS 100%, remainder of Ocimeae 97%). Isodon (BS 96%), Hyptidinae (BS 89%), and Plectranthinae (BS 92%) are supported as monophyletic groups within the Ocimeae. Although Ociminae forms a clade in the consensus tree there is less than BS 50% for this. The sistership of Ociminae and Plectranthinae is supported, though Ociminae has BS < 50% (Fig. 1A). The relative positions of the Hyptidinae, Isodon, and Ociminae plus Plectranthinae are unresolved, though Hyptidinae and Isodon are sisters in the EW analysis, but with BS < 50%. Within Ociminae the monophyly of Syncolostemon plus Hemizygia (BS 97%) and most species of Ocimum (BS 63%) are supported (Fig. 1B). Ocimum subgenus Nautochilus is supported (BS 78%) as a group separate from the remainder of Ocimum. The consensus tree supports the monophyly of Orthosiphon plus Fuerstia and Hoslundia. However, this grouping and also the resolution of relationships between named clades in Ociminae have BS < 50% (Fig. 1B). The monophyly of
Fig. 1. (AC) One most parsimonious tree after successive weighting of molecular data showing branch length (above branches) and bootstrap percentages (below branches, italicised, equal weights). Arrows indicate branches collapsing in the strict consensus trees with successive (unlled arrows) and equal (black arrows) weights. (A) Basal lineages; (B) Ociminae; (C) Plectranthinae.
284
Fig. 1. (continued)
Platostoma plus Basilicum and Haumaniastrum; and Endostemon plus Puntia is supported by the SW analysis only. Within Plectranthinae a clade comprising Pycnostachys, Holostylon, Anisochilus, and species of Plectranthus formerly placed in Coleus is supported (BS 97%) (Fig. 1C). Pycnostachys forms a monophyletic group within this Coleus clade (BS 96%). A clade which contains Plectranthus plus Tetradenia, Thorncroftia, and Aeollanthus is sister to the Coleus clade (BS < 50%).
Tetradenia plus Thorncroftia forms a clade with BS 100%. The Madagascan endemic genera Capitanopsis and Dauphinea are sisters (BS 76%) (Fig. 1C). The EW morphological analysis produced 760 trees with CI 0.31 and RI 0.85. There is little resolution in the strict consensus tree (data not shown). The SW morphological analysis produced 1960 trees with CI 0.31 and RI 0.85. No hard incongruences were found with the molecular MP trees (no contradictory clades supported by bootstrap >60%; data not shown).
285
Fig. 1. (continued)
Analyses combining morphological and molecular data again produced trees that showed no hard incongruences with the molecular MP trees (no contradictory clades supported by bootstrap > 60%; data not shown). Combined analyses showed less resolution than the molecular MP trees. 3.2. Bayesian phylogeny reconstruction The Bayesian phylogeny reconstruction of the three combined DNA partitions is shown in Fig. 2. Again the monophyly of the Ocimeae clade is supported (posterior probability (pp) 0.53) and Elsholtzia is supported as the sister group (pp 0.48). Lavandula is again supported as the sister group to the rest of the Ocimeae (pp 0.53) (Fig. 2A). Within the remainder of the Ocimeae there is no resolution of the relationships of major groups (Figs. 2A and B). However, the Hyptidinae clade (pp 0.84) and Isodon clade (pp 0.35) are supported (Fig. 2A). At lower level some groupings suggested by the EW MP analysis
are also supported: Ocimum minus subgenus Nautochilus (pp 0.95); Ocimum subgenus Nautochilus (pp 0.46) (Fig. 2B); and Tetradenia (pp 0.97); Thorncroftia (pp 0.99); Aeollanthus (pp 0.88); and Capitanopsis plus Dauphinea (pp 0.88) (Fig. 2C). Endostemon plus Puntia which is only supported by the SW, not EW, MP analysis is supported in the Bayesian reconstruction (pp 0.61, Fig. 2B). Several clades supported by the EW MP analysis are fragmented in the Bayesian reconstruction. The Orthosiphon, Coleus, and Plectranthus clades are all separated into two parts, whereas the Syncolostemon clade is fragmented into three (Figs. 2AC). The main areas of incongruence between the Bayesian and EW MP phylogeny reconstructions are caused by the fragmentation of the above clades in the Bayesian phylogeny inference (compare Figs. 1 and 2). However, these individual divisions of the fragmented clades produced by Bayesian inference are separated by branches showing low posterior probabilities (see Section 4).
286
The Bayesian analysis does not include gap characters. To try and assess the inuence of this on the recognition of clades, the distribution of gap characters was analysed and the results of a sample of clades is presented in Table 3. There appears to be no simple relationship between the occurrence of gap characters and the recognition of the clade in the Bayesian analysis. For example, the Coleus and Ocimum clades have similar branch lengths, proportion of gap characters dening them, and a similar proportion of informative gap characters within them. The Bayesian analysis supports the Ocimum clade, but not the Coleus clade (Figs. 2B and C). The MP analysis supports both, the Coleus clade having the higher BS (Table 3). 3.3. Optimisation of morphological and chemical characters on to the phylogeny Morphological and chemical characters, their states, consistency index (CI), retention index (RI), and re-
scaled consistency index (RC) after ACCTRAN optimisation on the MP EW tree shown in Fig. 3 are presented in Table 2. Only eight of the 43 chemical and morphological characters are not homoplastic. Spiral insertion of cymes is a synapomorphy for Pycnostachys within the Coleus clade, while cyme inserted on one side of the inorescence axis is a synapomorphy for Aeollanthus within the Plectranthus clade (character 6, Table 2). A hinged anterior lobe of the corolla is a synapomorphy for Hyptidinae (character 17, Table 2). Anterior stamen fusion is synapomorphic for Syncolostemon (character 22, Table 2). Disk lobes opposite the nutlets is a synapomorphy for Lavandula (character 30, Table 2), and dorsixed anthers are a synapomorphy for the Ocimeae (character 33, Table 2). One cell layer in the schlerenchyma region of the pericarp (character 34, Table 2) and bone-shaped cells of the schlerenchyma region (character 35, Table 2) are synapomorphies for Nepetoideae and 5,6-diOH-7,8-diOMe-Flavones
Fig. 2. (AC) Phylogenetic tree depicted from Bayesian inference and showing posterior probabilities above branches. (A) Basal lineages; (B) Ociminae plus Plectranthinae p.p.; (C) Plectranthinae p.p.
287
Fig. 2. (continued)
(character 41, Table 2) is an apomorphy for the clade comprising Nepeta, Thymus, Origanum, Clinopodium, and Mentha. Synthecous anthers are also a synapomorphy for Ocimeae (character 25, CI 0.66, Table 2). The CI is less than one due to changes among other states of this multistate character elsewhere in the analysis.
4. Discussion There is no well-supported incongruence between the two models of phylogeny reconstruction; however, it is noticeable that Bayesian reconstructions fragmented several clades to which both MP analyses and taxonomies gave support. Recently, Douady et al. (2003) and
288
Fig. 2. (continued)
Table 3 Table detailing support for selected clades Clade Proportion of characters which are gaps on dening branch/branch length in MP Analysis 0.4/17 0.7/7 0.28/7 0.28/7 0.75/4 No gaps/4 Proportion of informative characters within clade which are gaps in MP Analysis 0.5 0.41 0.33 0.30 0.39 0.41 Bayesian support (posterior probability) Bootstrap support EW MP analysis omitting gaps (%) 95 58 54 96 56 97 Bootstrap support EW MP analysis, gaps included (%) 89 96 63 97
Hyptidinae Isodon Ocimum Coleus Platostoma Syncolostemon
0.4 0.35 0.95

100
Alfaro et al. (2003) studied the link between the more traditional bootstrap analyses and Bayesian phylogeny inference using real data sets and simulations. They aim to shed light on the marked discrepancies [that] exist between non-parametric bootstrap proportions and Bayesian posterior probabilities, leading to diculties in the interpretation of sometimes strongly conicting results (Douady et al., 2003; p. 248). These studies found that the relation between Bayesian posterior probabilities and bootstrap percentages were highly variable and not interchangeable (Douady et al., 2003), although when a threshold value was chosen for accepting incorrect monophyletic group as true at 5%, all methods provided similar results in simulations (Alfaro et al.,
2003). As stated in Section 3, disagreement between Bayesian reconstructions and maximum parsimony mostly involved nodes with low posterior probabilities, tting well with the ndings of the studies mentioned above. Although the data matrices we used with both methods were not identical (gaps are not taken into account in Bayesian reconstructions), no clear pattern emerged here (Table 3), and we await further explorations of the behaviour of Bayesian reconstructions before making additional statements with regard to Ocimeae. Finally, we note that despite the fact that Bayesian reconstructions are sometimes described as representing much better estimates of phylogenetic accuracy than do nonparametric bootstrap (Wilcox
289
Fig. 3. (AC) Ultrametric NPRS Tree showing optimisation of the geographical distribution of terminal taxa and ACCTRAN optimisation of various characters. (A) Basal lineages; (B) Ociminae; (C) Plectranthinae. A relative time scale T to 4T has been added to allow easy reference between parts of the gure. Key to characters: 1 5-OH-6,7-diOMe-Flavones, absent ()), present (+); 2 5-OH-6,7,8-triOMe-Flavones absent ()), present (+); 3 5,6-diOH-7,8-diOMe-Flavones absent ()), present (+), equivocal (?); Q quinonoid diterpenoids absent ()), present (+); D declinate stamens absent ()), present (+), stamens spreading (s); H hinge of anterior corolla lobe absent ()), present (+). F fusion of all stamens absent ()), present (+); A fusion of anterior stamens absent ()), present (+); S sigmoid corolla tube absent ()), present (+). C (in (C) only) anterior corolla lobe deexed present (+), absent unless marked.
et al., 2002, p. 361), Bayesian analysis can be excessively liberal when concatenated gene sequences are used (Suzuki and Glazko, 2002, p. 16138) arguing for increased condence in the conservative boostrap. The topology derived from the molecular EW MP analysis is used as the basis for the discussion below. The SW analysis is referred to when currently recognised taxa
are not supported by the EW analysis due to the lack of resolution, but are supported by the SW analysis (Fig. 1). 4.1. Phylogeny and taxonomy Tribe Ocimeae is a well supported monophyletic group with a morphological synapomorphy of dorsixed,
290
Fig. 3. (continued)
synthecous anthers. Tribe Elsholtzieae is sister to the Ocimeae. This is in agreement with plastid DNA restriction site studies of Wagsta et al. (1998), but not the rbcL study of Kaufmann and Wink (1994) where Ocimum is embedded within the tribe Mentheae, and is sister to Melissa and Origanum [Majorana]. Subtribe Lavandulinae, which can be diagnosed by the synapomorphy of having disk lobes opposite the nutlets, is sister to the remainder of the Ocimeae. Examination of
pollen characters also suggests the presence of spinulose elements on the aperture membrane as a putative synapomorphy for Lavandulinae. The relationships among Isodon, Hyptidinae, Plectranthinae, and Ociminae are unresolved. The analysis indicates that only the Hyptidinae has a clear non-molecular synapomorphy of a hinged anterior corolla lip. However, Asterohyptis, not included in this analysis, but normally placed within the Hyptidinae (Ryding, 1992)
291
Fig. 3. (continued)
lacks this character. Isodon does not clearly belong in any of the existing subtribes and it is recognised in Harley and Paton (2003) and Harley et al. (in press) along with putative relatives Hanceola and Siphocranion as subtribe Hanceolinae. Several currently recognised genera are not monophyletic. Within Ocimineae, Syncolostemon is monophyletic with the inclusion of Hemizygia. This clade can be diagnosed by the synapomorphy of fused anterior stamens (character 22, Table 2). The pollen of this clade
provides putative synapomorphies. The primary reticulum lumina are large. The muri are continuous and tend to form a continuous undulated margin around the colpus. The secondary reticulum which is monoplanar and continuous, is reticulate. The muri form a very ne delicate net which, surprisingly, tends to survive acetolysis intact, the lumina average upwards of 1030 or more in each primary lumen. These genera should probably be merged under the earliest name Syncolostemon. This will be explored in more depth by one of us
292
(D.O.) in a separate paper. Although the relationships between Ocimum and Nautochilus are unresolved in the EW analysis, Ocimum as currently recognised (Paton, 1997) is polyphyletic on the basis of the SW analysis. Separate recognition of the monophyletic subgenus Nautochilus and the monophyletic remainder of Ocimum produces two monophyletic groups (BS 78 and 63%, respectively), though neither has clear non-molecular synapomorphies (Fig. 1B). Generic recognition of Nautochilus, following Bremekamp (1933), needs to be considered in more detail. Orthosiphon is monophyletic with the inclusion of Fuerstia and Hoslundia, but the Orthosiphon clade lacks BS (<50%) and clear non-molecular synapomorphies. It may be better to merge these taxa under the earliest name Hoslundia Vahl. However, as this will cause many changes in existing nomenclature, we prefer to wait until further evidence for this relationship is available. The currently recognised Platostoma (Paton, 1997) is not supported by the EW anaysis, the relationships of the species being unresolved. The SW analysis suggests that Platostoma would be monophyletic with the inclusion of Basilicum and Haumaniastrum. Further work is needed to explore this relationship. A close relationship between Puntia and Endostemon is also suggested by the SW MP analysis, Baysian inference and morphology including corolla, stamen, style, and pericarp characters. The merging of Puntia into Endostemon is discussed in more detail by Ryding et al. (in press). Within Plectranthinae, there are two clades: Coleus and Plectranthus (Fig. 1C). The Coleus clade contains many species of Plectranthus sometimes recognised as Coleus (Li and Hedge, 1994). Neither the Plectranthus nor Coleus clades can be diagnosed by non-molecular synapomorphies. A few subclades of Plectranthinae are diagnosable by non-molecular synapomorphies. The analysis indicates that spirally inserted cymes is a synapomorphy for Pycnostachys, but Plectranthus longipetiolatus, not included in this analysis, also displays this character. Cymes inserted on one side of the inorescence axis is a synapomorphy for Aeollanthus. Aeollanthus also has an apical position of the lumen of the bone cells and crystals in the schlerenchyma region of the pericarp. Apical lumina are found in Ocimum and Syncolostemon clades of Ociminae, while crystals are found throughout Ociminae and Hyptidinae. However, these characters do not occur elsewhere in Plectranthinae. Some other clades have synapomorphies when viewed only in the context of Plectranthinae. Tetradenia and Thorncroftia have free lateral lobes of the corolla. However, this character occurs elsewhere within Tribe Ocimeae, e.g., Lavandula, Endostemon, and Puntia. Plectranthus as currently recognised is paraphyletic. Further sampling within the Plectranthus and Coleus clades is necessary to investigate how better to recognise monophyletic groups within Plectranthinae.
Within subtribe Hyptidinae, Hyptis is paraphyletic, but this clade is under-represented in this analysis and increased sampling is required before conclusions are drawn. Generally the existing placement of genera within subtribes based on morphology and pericarp anatomy (Ryding, 1992, 1993, 1995) is supported by the molecular phylogenetic analysis. There are two exceptions: (1) Tetradenia which is similar in morphology to subtribe Ociminae, but which Ryding (1993) suggested had an isolated position within tribe Ocimeae on the basis of pericarp anatomy, belongs in the Plectranthinae clade in the molecular MP analysis. (2) Basilicum, placed in the Orthosiphon Group group by Ryding, is placed in the Platostoma clade in the molecular SW MP analysis, though its position is unresolved in the EW analysis. Both Tetradenia and Basilicum taxa have much reduced owers (ca. 3 mm long) and lack morphological characters shared by their nearest relatives on the basis of the molecular study. Within the study group, morphology is not a reliable indicator of phylogeny in situations where there is a reduction in ower size. 4.2. Floral evolution and pollination Ocimeae are usually considered to have stamens pressed against the anterior, or lower side of the ower (declinate) and deliver pollen to the ventral surface of visitors (sternotribic) (Huck, 1992; Van Der Pijl, 1972). Other members of subfamily Nepetoideae have stamens ascending under the posterior (upper) lip of the corolla and pollen is deposited on the back of pollinators (nototribic) (Van Der Pijl, 1972). Pollinators of Ocimeae include bees, butteries, and ies (Huck, 1992; Nilsson et al., 1985; Potgeiter et al., 1999; Van Der Pijl, 1972). Declinate stamens are not a synapomorphy for the Ocimeae (Figs. 3AC): stamens are spreading in Platostoma caeruleum and in Tetradenia, the posterior pair directed upwards and the anterior pair directed downwards. This change from declinate stamens has independently occurred in these species. Apart from this change, the position of the stamens relative to the corolla lobes does seem to be constrained in that stamens are never ascending under the posterior corolla lobes as in the remainder of the Nepetoideae (Fig. 3A). However, nototribic pollination does occur in some species of Hypenia (Hyptidinae), but this is achieved by the resupination of the whole corolla (Atkinson, 1998), rather than a change in stamen position relative to the corolla lobes. There are several adaptations to pollinator behaviour within Ocimeae. The anterior corolla lip of the corolla can provide a landing platform. In Hyptidinae the stamens are held under tension within the anterior lip which is hinged. Upon landing of an insect, usually bees, the stamens are released explosively shedding pollen
293
from the anthers (Harley, 1971). A similar explosive mechanism has independently evolved in some species of Aeollanthus (Hedge, 1972; Ryding, 1981). These species lack the synapomorphic hinge of the Hyptidinae (Fig. 3A), the triggering in this case involves the apex of the anterior lip which is hooded and holds the lip in place until triggered. Van Der Pijl (1972) suggests that staminal fusion seen in the Coleus clade provides a stronger landing platform than would free stamens. Fused stamens have evolved independently in the Syncolostemon clade (anterior pair only, Fig. 3B) and in the Coleus clade (all stamens, Fig. 3C). The fusion is not consistent within these clades. Although anterior staminal fusion is synapomorphic for the Syncolostemon clade, there is much variation in the degree of stamen fusion from almost complete fusion at the base only. Staminal fusion is lost several times within the Coleus clade, e.g., Plectranthus cylindraceus and P. sanguineus (Fig. 3C). Nilsson et al. (1985) suggest that a lack of a rm landing platform in Plectranthus vestitus, which has a deexed anterior corolla lobe, favours hovering insects. Deexed anterior corolla lobes occur many times in Ocimeae (CI 0.04, Table 2; Fig. 3C, character only mapped within Plectranthinae), and within the southern African Plectranthinae this may be associated with long-tongued hovering insects whose behaviour does not require a landing platform (Nilsson et al., 1985). Deexed lateral corolla lobes seen in Thorncroftia may also be associated with a reduction in landing platform. However, deexed anterior and free lateral corolla lobes are also found in Lavandula, Endostemon, Puntia, and Tetradenia. In Lavandula, Tetradenia, and Platostoma caeruleum, the inorescence is condensed. The close proximity of the corollas and the spreading deexed corolla lobes together present a rm landing platform for insects, such as in bees visiting Lavandula. In Endostemon and Puntia, however, the inorescence is not condensed. Sigmoid corolla tubes are found in all the Coleus clade and in Plectranthus laxiorus/P. petiolaris subclade of the Plectranthus clade (Fig. 3C). The sigmoid tube is always associated with a horizontal anterior corolla lobe (Fig. 3C). This combination of characters will favour landing insects with exible probosci. The need for increased sampling and the lack of supported resolution at the base of the Plectranthus clade, make it impossible to draw rm conclusions about the direction of evolution between straight tubes with deexed lobes suiting hovering pollinators, and sigmoid tubes with horizontal anterior lips suiting landing, exible tongued insects. Synthecous, dorsixed anthers are synapomorphies for Ocimeae (Fig. 3A). Generally anther shape is disk-like and varies little in size and shape. The anther is comprised of two thecae which split along their length and shed their contents into a shared central area. However, in Holostylon, a group of ve species, the lower parts of the thecae
do not completely split forming a pouch and the upper parts split to form a pore. Similar anther structure is also seen in Plectranthus edulis and P. punctatus, but in this case the anther pore is central rather than apical. These species were not included in the analyses, but they are morphologically most similar to species of the Coleus clade which also includes Holostylon. The style in Holostylon is entire, with a small depression at the apex, rather than bid as in the remainder of the Plectranthinae. This character is not seen elsewhere in the Ocimeae. The combination of porose anthers and a depression or chamber in the style is seen in buzz pollinated Cassia (Dulberger et al., 1994; Gottsberger and Silberbauer-Gottsberger, 1988). Gottsberger and Silberbauer-Gottsberger (1988) suggest that vibration of the stamens and dispersal of the pollen is made more ecient by the rigidity of the stamens. Rigidity is increased by staminal fusion in Holostylon. Gottsberger and Silberbauer-Gottsberger (1988) also suggest that the corolla lobe, which is folded around the stamens of Chamaecrista species, acts to accurately channel pollen onto the pollinators body. The deeply cucullate anterior corolla lobe of Holostylon may perform the same function. Buzz pollination has not been recorded in the Lamiaceae and eld work is needed to establish if it does occur in Holostylon. 4.3. Phytochemistry Overall, the quinone diterpenoids and various types of avonoids in the leaf exudates dier in their distributions across the phylogeny (Fig. 3). For example, in Plectranthinae there are no species with 8-oxygenated avones; they only contain 5-hydroxy-6,7-dimethoxyflavones (Fig. 3C). In contrast, both 5-hydroxy-6,7-dimethoxyavones and 8-oxygenated avones were present in all species belonging to the Ocimum clade (Fig. 3B). The prole of avones of the genera Endostemon and Basilicum diered from Ocimum (Fig. 3B). For example, Endostemon obtusifolius and Basilicum polystachyon contain 5-hydroxy-6,7-methoxyavones but no 8-oxygenated avones. They also contain avonols which are absent in Ocimum. Diterpenoids are produced by many species belonging to most subfamilies of the Lamiaceae (Vestri Alvatenga et al., 2001), but the occurrence of diterpenoids with a quinone group is largely restricted to genera in subfamily Nepetoideae (Hegnauer, 1989). So far no diterpenoids have been reported from species of Ocimum, whereas they have been reported from many species of Plectranthus (Hegnauer, 1989). All species investigated chemically that belong to the Plectranthus clade (Fig. 3C) produce avones and lack quinonoid diterpenoids (although they may produce dierent types of diterpenoids), whereas most species of the Coleus clade produce quinonoid diterpenoids and lack
294
avonoids. Exceptions are P. cylindraceus, P. buchananii, and Holostylon katagense, which belong to the Coleus clade (Fig. 3C). The latter two species produce both quinonoid diterpenoids and avones, whereas P. cylindraceus produces neither, but it does contain a wide range of avonols. These species with aberrent chemistry form a subclade within the Coleus clade in the SW analysis (Fig. 1C) but not in the EW analysis (Fig. 3C). These chemical dierences within Ocimeae could contribute to dierences in the recorded medicinal as well as pesticidal uses of the species in the dierent clades. For example, the diterpenoids contribute to the anti-microbial (Rijo et al., 2002) and anti-oxidant (Narukawa et al., 2001) activity of Plectranthus, whereas the avonoids contribute to the anti-oxidant activity of Ocimum (Devi et al., 2000). This new phylogeny (Fig. 1) has provided a framework to select species for biological studies. For example, are the anti-cancer activity of diterpenoids isolated from species of Plectranthus (Marques et al., 2002) distributed among clades or are they restricted to species within a clade? Mapping on the phylogenetic tree the distribution of diterpenoids, such as coleon U, along with the known medicinal uses of species of Plectranthus could assist the selection of plants for further anti-cancer screens. 4.4. Biogeography Due to the uncertainty surrounding dates of the earliest fossil hexacolpate pollen (Harley et al., in press), and the paucity of sampling of Nepetoideae and potential sister taxa within Viticoideae, the relative time of the NPRS tree was not transformed into absolute ages as no clear calibration point could be identied. The conclusions below should only be regarded as tentative as only 12% of species are covered in the analysis. Optimisation of geography on the NPRS tree indicates older nodes of Ocimeae occurring in Asia (Fig. 3A). It should be noted that Hanceola and Siphocranion, likely relatives of Isodon, but absent from this analysis, are also Asian (Paton and Ryding, 1998). The clade comprising Ociminae and Plectranthinae suggests an arrival in Africa at a later relative time (Fig. 3A). Within this basally African clade there are several secondary arrivals in Asia. Such arrivals in the Platosoma, Orthosiphon, and in the Plectranthus albicalyx/heleri and Anisochilus/P. parisii clades all occurred more recently (Figs. 3B and C). The secondary arrival of P. scutellarioides in Asia is even later (Fig. 3C). More recent migrations between Africa and Asia are also likely as several species of Ocimum and Plectranthus are currently found both in Tropical Africa and Asia, for example, P. caninus Roth and O. americanum L. Several species of Ocimum and a few of Hyptis are pan-tropical weeds. Colonisation of Madagascar occurred at least twice in the Plectranthinae clade (Fig. 3C): rst in Capitanopsis/
Dauphinea and then in Tetradenia (Fig. 3C). However, these clades need to be more intensively sampled before rm conclusions are drawn. The Capitanopsis/Dauphinea clade, endemic to Madagascar, probably also includes Madlabium and comprises six species of Madagascan Plectranthus or Pectranthus, whereas Tetradenia has 12 species in Madagascar and three in Africa. Orthosiphon, Syncolostemon, and Platostoma are all found in Madagascar, Tropical Africa, and Asia, but unfortunately sequence data from Madagascan members of these clades are not available. However, this suggests at least ve arrivals of Ocimeae in Madagascar in geological time. Plectranthus luteus and P. tettensis and Pycnostachys caerulea, all likely to t into the Coleus clade, and Endostemon tenuiorum are all found both in Tropical Africa and Madagascar suggesting more recent movement between Madagascar and Africa. Tribe Ocimeae is mainly represented in the New World by subtribe Hyptidinae comprising around 380 species. Ocimum has ve species native to the New World and Catoferia has only three species. Hyptidinae represents the earliest arrival in the New World from Asia in (Fig. 3A). Catoferia represents a later occurrence in the New World, possibly evolving from an African ancestor (Fig. 3B). However, resolution in the Platostoma clade is poor with BS < 50% (Fig. 1B) and a radiation from Asia cannot be discounted. Ocimum selloi represents an even later movement to the New World (Fig. 3B). Thus there are at least three separate arrivals of the Ocimeae in the New World. There are a few species which occur both in the New and Old World, for example, Ocimum gratissimum, O. basilicum, and Plectranthus neochilus. These species are widely used medicinally and horticulturally and their distribution is heavily inuenced by this. In addition to major distributions in Asia, Tropical Africa, and the New World Tropics, Ocimeae are also found in Australia, and the Mediterranean and Macaronesian regions. Tribe Ocimeae is represented in Australia by some members of the Coleus clade and some introduced species. However, these species of the Coleus clade were not included in the analysis. Lavandula is found in the Mediterranean and Macaronesia, but also in Arabia and India. Arrival of Lavandulinae in the Mediterranean and Macaronesia is likely to have been from Asian ancestor, rather than from tropical Africa (Fig. 3A).
Acknowledgments This study was partially supported by the BAT Biodiversity Partnership with RBG Kew. We thank Her Royal Highness Princess Mahachakri Sirindorn of Thailand for supporting the work of Somran Suddee and the University of the Witwatersrand, the Mellon Foundation Fund, and the Lennox Boyd Fund, Kew for
295
supporting Donald Otieno. We thank Richard Olmstead for sending DNA of Elsholtzia, Melissa, Tectona, and Congea. We also thank Kevin Balkwill for collecting Appendix A
material of Thorncroftia. We thank Olof Ryding for discussion on scoring of pericarp characters and other helpful comments.
Data matrix used for mapping morphological, chemical, and distribution characters onto phylogenetic trees. Character order follows Table 2. A (01) B (12) C (13) D (23) Aeollanthus buchnerianus Aeollanthus densiorus Alvesia rosmarinifolia Anisochilus harmandii Anisochilus pallidus Basilicum polystachyon Callicarpa americana Callicarpa japonica Capitanopsis angustifolia Catoferia chiapensis Clinopodium vulgare Congea tomentosa Dauphinea brevilabra Elsholtzia stauntonii Endostemon obtusifolius Fuerstia africana Gmelina hystrix Haumaniastrum katangense Hemizygia albiora Hemizygia foliosa Hemizygia incana Hemizygia modesta Hemizygia obermeyerae Hemizygia parvifolia Hemizygia persimilis Hemizygia pretoriae Hemizygia punctata Hemizygia stalmansii Hemizygia subvelutina Hemizygia teucriifolia Hemizygia transvaalensis Hoslundia opposita Hypenia brachystachys Hypenia macrantha Hypenia sp. Hyptis brevipes1 Hyptis brevipes2 Hyptis capitata Hyptis eriocephala Hyptis oribunda Hyptis leptostachys ssp caatingense Hyptis suaveolens Isodon coetsa Isodon hispidus Isodon lophanthoides Isodon pharicus Isodon rugosus 00201 00201 002C1 00231 00231 00211 01030 01030 01221 00231 00130 01?30 00221 00130 002C1 00201 01010 10221 00211 00201 00211 00211 00211 00201 00211 00211 00201 00201 00211 00201 00211 00201 01210 01200 01200 01130 01130 01130 01130 001C0 00130 00130 01030 01030 01030 01030 01030 20011 20011 00004 00100 00100 00000 00000 00000 00000 02003 00001 00000 00003 00000 00000 00000 00000 00002 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 A0100 00000 00000 01001 01000 01000 01000 01000 01000 01000 00000 00000 00000 0000A 00001 00001 00000 00001 03100 03000 04200 00001 20001 01000 00010 00010 00200 15010 01020 00010 10200 00010 00010 01000 00010 10100 01001 01001 01001 01001 01001 01001 01001 01000 01000 01001 01001 00000 01001 01000 00010 00010 00010 01010 01010 01010 01010 00010 01010 01010 00100 00000 00000 00000 00100 10111 10011 10011 20011 20011 00011 00122 00122 10111 00011 10101 00122 10111 00021 00111 00011 00121 00011 00011 00011 10111 10111 00011 00011 00111 00111 00111 00011 00111 20011 00111 00011 01111 11111 11111 01111 01111 01111 01111 01111 01111 01111 10011 00011 00011 10011 00011 00201 00201 00201 00201 00201 00101 00002 00002 00201 00101 00100 00102 00201 00100 00301 00101 00002 00021 01001 01001 01001 01A01 01001 01001 01A01 01A01 01001 01001 01001 10201 01001 00101 00101 00101 00101 00101 00101 00101 00101 00101 00101 00101 00001 00001 00001 00001 00001 00010 00010 00010 00010 00010 10000 00000 00000 00010 30010 40000 00000 00010 00010 10100 10010 40000 00010 00010 00010 00010 00010 00010 10010 00010 00010 00010 00010 00010 30010 00010 10010 00010 00010 00010 00000 00000 00000 00000 00000 10000 00000 00000 00000 00000 00000 00000 10101 10101 10101 10101 10101 10101 21010 21010 101?? 101?? 000?? 210AA 101?? 10001 10101 10101 21010 101?? 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 00101 00101 00101 00101 00101 00101 00101 00101 00101 00101 10101 10101 10101 10101 10101 11110 11110 000?? 010?? 010?? 10?10 102?? 102?? ??? ??? ??? 100?? ???10 000?? 10?10 10??? 102?? ??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 01010 10??? 10??? 100?? 100?? 100?? 100?? 100?? 100?? 100?? 100?? 100?? 100?? 000?? 000?? 000?? 000?? 000?? 0000 0100 ???0 ???2 ???2 010E ???6 ???2 ???1 ???6 1??3 ???2 0001 ???2 0100 ???0 ???2 ???0 ???0 ???0 ???0 ???0 ???0 ???0 ???0 ???0 ???0 ???0 ???0 0010 ???0 ???0 ???6 ???6 ???6 ???6 ???6 ???6 ???6 ???6 ???6 ???6 ???2 ???2 ???2 ???3 ???3
296
Appendix A (continued) Isodon ternifolius Lavandula buchii Lavandula maroccana Lavandula minutolii Lavandula rotundifolia Mellissa ocinalis Mentha suaveolens Nepeta ssa Nepeta menthoides Nepeta racemosa Nepeta straussii Ocimum americanum var pilosum Ocimum basilicum Ocimum citriodorum Ocimum lamentosum Ocimum gratissimum var gratissimum Ocimum gratissimum var macrophyllum1 Ocimum gratissimum var macrophyllum2 Ocimum labiatum1 Ocimum labiatum2 Ocimum selloi Ocimum serratum Ocimum tenuiorum Origanum vulgare Orthosiphon aristatus Orthosiphon parishii Orthosiphon rotundifolius Orthosiphon rubicundus Platostoma africanum Platostoma annamense Platostoma calcaratum var garettii Platostoma cambodgense var cambodgense Platostoma cambodgense var subulatum Platostoma cochinchinense Platostoma coeruleum Platostoma coloratum var coloratum Platostoma coloratum var minimum Platostoma mbriatum Platostoma hildebrandtii Platostoma hispidum Platostoma intermedium Platostoma kerrii Platostoma mekongense Platostoma ocimoides Platostoma rotundifolium Platostoma rubrum Platostoma siamense Platostoma tectum Plectranthus albicalyx Plectranthus alboviolaceus Plectranthus amboinicus Plectranthus barbatus1 Plectranthus barbatus2 01030 00201 00201 00201 00201 011C0 001D0 01130 01130 01130 01130 00221 00221 00221 00221 00221 00221 00221 00211 00211 00221 00211 00231 00200 002C1 00231 00231 00211 10231 00231 10231 10231 10231 10231 10131 10131 10131 10231 10231 10231 102C1 10211 10231 102C1 10131 10131 10131 10131 00221 00211 00131 00131 00131 00000 01001 01001 01001 01001 00001 00000 00001 00001 00001 00001 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00001 00001 00001 00001 00001 00001 00001 00001 00001 00001 00001 00000 00001 00001 00001 00001 00001 00001 00001 00001 00100 00003 00100 00100 00100 00100 00010 00010 00010 00010 01010 00010 20010 00011 00011 00011 00000 00000 00000 01000 10000 10000 10000 00000 00000 00000 00000 00000 00000 11000 01000 11000 01000 15100 15300 15300 15100 15100 15100 15100 10100 10100 15100 10100 00000 15300 15300 15100 15300 15100 15000 10100 15100 10001 01200 00001 00001 00001 10011 00111 00111 00111 00111 00001 00021 10000 10000 10000 10000 00011 00011 00011 00011 00011 00011 00011 10111 10111 00011 10111 00011 00001 00011 00011 00011 00011 10011 10111 10111 10011 10011 10011 10021 10011 10011 10011 10011 10011 10011 10011 10011 10011 10011 00011 10011 10011 20011 10011 20011 20011 20011 00001 00101 00101 00101 00101 00100 00100 00300 00300 00300 00300 00031 00031 00031 00031 00031 00031 00031 00001 00001 00031 00001 00001 00100 00101 00101 00101 00101 00021 00021 00021 00041 00041 00041 00021 00021 00021 00041 00021 00021 00021 00021 00021 00021 00021 00041 00021 00041 10201 20201 10201 10201 10201 00000 10001 10001 10001 10001 00100 00000 00010 00010 00010 00010 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 10010 10010 10010 10010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 10101 10101 10101 10101 101?1 100?? 00001 00001 00001 00001 00001 10101 10101 10101 10101 101?? 101?? 101?? 10101 10101 101?? 10101 10101 100?? 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 000?? 00011 00011 00011 00011 ??? 100?? A0011 A0011 A0011 A0011 10?11 10?11 10?11 10??? ???11 ??? ??? 101?? 101?? ??? 101?? 10111 ??? 100?? 100?? 100?? 100?? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 10??? 0?0?? 0?010 010?? 01000 0?000 ???2 0004 0003 0004 0004 ???3 1??3 1003 1003 1003 1003 0002 000D 000D ???D 000D ???D ???D ???0 ???0 ???6 ???0 0002 1??3 ???2 ???2 ???2 ???2 ???0 ???2 ???2 ???2 ???2 ???2 ???0 ???2 ???2 ???2 ???0 ???2 ???2 ???2 ???2 ???2 ???0 ???2 ???0 ???2 ???2 0000 ???0 0010 0010
297
Appendix A (continued) Plectranthus buchananii Plectranthus calycinus Plectranthus ciliatus Plectranthus coeruleus Plectranthus crassus Plectranthus cylindraceus Plectranthus fredricii Plectranthus heleri1 Plectranthus heleri2 Plectranthus glabratus Plectranthus laxiorus Plectranthus oertendahlii Plectranthus parishii Plectranthus petiolaris Plectranthus puberulentus2 Plectranthus puberulentus1 Plectranthus sanguineus Plectranthus scutellaroides Plectranthus thyrsoideus Plectranthus xerophilus Prostanthera nivea Prostanthera petrophila Puntia stenocaulis Pycnostachys reticulata Pycnostachys umbrosa Pycnostachys urticifolia Rosmarinus ocinalis Salvia guaranitica Syncolostemon argenteus Syncolostemon comptonii Syncolostemon abellifolius Syncolostemon macranthus Syncolostemon parviorus Syncolostemon rotundifolius Tectona grandis Tetradenia fruticosa Tetradenia nervosa Thorncroftia longifolia Thorncroftia media Thymus serphyllum var citriodorum Vitex trifolium References
Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov Chain Monte Carlo sampling and bootstrapping in assessing phylogenetic condence. Mol. Biol. Evol. 20, 255266. Atkinson, R., 1998. A taxonomic revision of Hypenia (Mart. ex Benth.) Harley (Labiatae). Unpublished Ph.D. Dissertation. University of St. Andrews, Scotland. Bremekamp, C.E.B., 1933. Nautochilus, Ann. Transv. Mus. 15, 253. Cantino, P.D., Harley, R.M., Wagsta, S.J., 1992. Genera of Labiatae: Status and Classication. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, pp. 502507.
00131 01131 00211 00221 00131 00131 01131 00211 00211 00131 01221 00211 01131 01221 00131 00131 00131 0A131 00131 00131 01200 01200 00201 00201 00201 00201 00201 00131 00131 00201 00201 00201 00201 00201 01030 00211 00211 00201 00201 00130 010C0
00100 00100 00003 00010 00100 00100 00100 00100 00100 00100 00000 00003 00100 00000 00100 00100 00100 00100 00100 00100 00002 00002 00000 11?00 11?00 11?00 00002 00001 00001 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 00001 00000
00001 00200 10200 00001 20001 20001 00001 00001 00001 00001 00000 10200 00001 00000 00001 00001 20001 10001 00001 00001 15120 15120 00010 02001 02001 02001 00120 00020 00000 01001 00000 01000 01000 01000 00010 20010 20010 00010 00010 00020 00010
20011 20011 00111 B0011 20011 20011 20011 20011 20011 20011 20011 10011 20011 20011 20011 20011 20011 20011 20011 20011 00021 00021 00011 20011 20011 20011 00102 20102 00111 10111 00011 10111 00011 00111 00122 00121 00121 00111 00111 00001 00021
10201 00201 00201 10201 10201 00201 10201 10201 10201 00201 00201 00201 00201 00201 10201 10201 00201 10201 10201 10201 00000 00000 00301 10201 10201 10201 00??? 00??? 01001 01001 01001 01001 01001 01001 00102 00101 00101 00201 00201 00100 00002
00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 00010 02000 02000 00100 00010 00010 00010 40000 40010 00010 00010 00010 00010 00010 00010 02000 00010 00010 00010 00010 00000 00000
10101 10101 101?? 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 10101 00010 00010 101?? 10101 10101 101?1 000?? 10001 10101 10101 10101 10101 10101 10101 210?? 10101 10101 10101 10101 100?? 21010
0?010 0?0?? ???10 0?000 0?000 01000 00000 0?0?? 0?0?? 0?0?? 000?? 01010 0?0?? 0?010 0?0?? 0?0?? 0?000 0?0?? 0???? 01000 102?? 102?? ??? 010?? 01000 010?? ??? 000?? 101?? 101?? 101?? 101?? 101?? 101?? ??? 000?? 00010 000?? 00010 ???11 102??
0010 ???0 0000 001A 0010 0100 0010 ???2 ???2 ???2 ???0 0000 ???2 0000 ???0 ???0 0010 ???2 ???0 0010 ???5 ???5 ???0 ???0 0010 ???0 ???3 ???6 ???0 ???0 ???0 ???0 ???0 ???0 ???2 ???1 0001 ???0 0000 1003 ???2
Cox, A.V., 1997. Paupgap Version 1.12.Program and Documentation. Royal Botanic Gardens, Kew. Devi, P.U., Ganasoundari, A., Vrinda, B., Srinivasan, K.K., Unnikrishnan, M.K., 2000. Radiation protection by the Ocimum avonoids orientin and vicenin: mechanisms of action. Radiat. Res. 154, 455460. Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J.P., 2003. Comparison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability. Mol. Biol. Evol. 20, 248254. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytoch. Bull. 19, 1115. Dulberger, R., Smith, M.B., Bawa, K.S., 1994. The stigmatic orice in Cassia, Senna and Chamaecrista (Caesalpiniaceae): morphological
298
A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299 Maddison, W.P., Maddison, D.R., 1999. MacClade, Version 3.08, Analysis of Phylogeny and Character Evolution. Sinauer Associates Inc, Sunderland, MA, USA. Marin, P.D., Grayer, R.J., Kite, G.C., Matevski, V., 2003. External leaf avonoids of Thymus species from Macedonia. Biochem. Syst. Ecol. 31, 12911307. Marques, C.G., Pedro, M., Simoes, M.F.A., Nascimento, M.S.J., Pinto, M.M.M., Rodriquez, B., 2002. Eect of abietane diterpenes from Plectranthus grandidentatus on the growth of human cancer cell lines. Planta Med. 68, 839840. Morhy, L., Gomes, J., Laboriau, J.G., 1970. Ocimum nudicaule Benth., A new source of methylchavicol. Ann. Acad. Bras. Cienc. 42, 147 158. Nilsson, L.A., Jonsson, L., Rason, L.E., Randrianjohany, E., 1985. Pollination of Plectranthus vestitus by trap-lining hovering bees in Madagascar. Pl. Syst. Evol. 150, 223226. Narukawa, Y., Shimizu, N., Shimotohno, K., et al., 2001. Two new diterpenoids from Plectranthus nummularius. Briq. Chem. Pharm. Bull., 11821184. Oxelman, B., Liden, M., Berglund, D., 1997. Chloroplast rps16 intron Phylogeny of the Tribe Sileneae (Caryophyllaceae). Pl. Syst. Evol. 206, 393410. Paton, A.J., 1997. Classication and species of Platostoma and its relationship with Haumaniastrum (Labiatae). Kew Bull. 52, 257 292. Paton, A., Harley, R.M., Harley, M.M., 1999. Ocimuman overview of relationships and classication. In: Holm, Y., Hiltunen R. (Eds) Ocimum. In: Hardman (Ed.), Medicinal and Aromatic Plants Industrial Proles. Harwood Academic, Amsterdam, pp. 138. Paton, A., Ryding, O., 1998. Hanceola, Siphocranion and Isodon and their position in the Ocimeae (Labiatae). Kew Bull. 53, 723731. Potgeiter, C.J., Edwards, T.J., Miller, R.M., Van Staden, J., 1999. Pollination of seven Plectranthus spp. (Lamiaceae) in southern Natal, South Africa. Pl. Syst. Evol. 218, 99112. Rijo, P., Gaspar-Marques, C., Simoes, M.F., Duarte, A., Apreda-Ojas, M.D., Cano, F.H., Rodriguez, B., 2002. Neoclerodane and labdane diterpenoids from Plectranthus ornatus. J. Nat. Prod. 65, 1387 1390. ~ez, D., Obo n De Castro, C., 1992. The ethnobotany of Riviera-Nun Old World Labiatae. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, pp. 455473. Ryding, O., 1981. The Aeollanthus buchnerianus group (Labiatae). Nord. J. Bot. 1, 154164. Ryding, O., 1992. Pericarp structure and phylogeny within Lamiaceae Subfamily Nepetoideae Tribe Ocimeae. Nord. J. Bot. 12, 273298. Ryding, O., 1993. Pericarp structure and systematic positions of ve genera of Lamiaceae Subfamily Nepetoideae Tribe Ocimeae. Nord. J. Bot. 13, 631635. Ryding, O., 1995. Pericarp structure and phylogeny of the Lamiaceae Verbenaceae complex. Plant Syst. Evol. 198, 101141. Ryding, O., Paton, A., Thulin, M., Springate, D., in press. Reconsideration of the genus Puntia and a new species of the genus Endostemon (Lamiaceae). Kew Bull. Sanderson, M.J., 1997. A nonparametric approach to estimating divergence times in the absence of rate constancy. Mol. Biol. Evol. 14, 12181231. Suzuki, Y., Glazko, G.V., Nei, M., 2002. Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proc. Natl. Acad. Sci. USA 99, 1613816143. Swoord, D.L., 2001. Paup* 4.0: Phylogenetic Analysis Using Parsimony (* and other methods). Sinauer Associates, Sunderland, MA. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991. Universal primers for amplication of three non-coding regions of chloroplast DNA. Pl. Mol. Biol. 17, 11051109. Van Der Pijl, L., 1972. Functional considerations and observations on the owers of some Labiatae. Blumea 20, 93103.
variation, function during pollination, and possible adaptive signicance. Am. J. Bot. 81, 13901396. Elden as, P., Linder, H.P., 2000. Congruence and complementarity of morphological and trnl-f sequence data and the phylogeny of African Restionaceae. Syst. Bot. 25, 692707. Farris, J.S., 1969. A successive approximations approach to character weighting. Syst. Zool. 18, 274385. Farris, J.S., 1989. The retention index and the rescaled consistency index. Cladistics 5, 417419. Felsenstein, J., 1985. Condence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783791. Fitch, W.M., 1971. Toward dening the course of evolution: minimum change for a specic tree topology. Syst. Zool. 20, 406416. Githinji, C.W., Kokwaro, J.O., 1993. Ethnomedicinal study of major species in the family Labiatae from Kenya. J. Ethnopharm. 39, 197203. Grayer, R.J., Bryan, S.E., Veitch, N.C., Goldstone, F.J., Paton, A., Wollenweber, E., 1996. External avones in sweet basil, Ocimum basilicum, and related taxa. Phytochemistry 43, 1041 1047. Grayer, R.J., Veitch, N.C., Kite, G.C., Price, A.M., Kokubun, T., 2001. Distribution of 8-oxygenated leaf-surface avones in Ocimum. Phytochemistry 56, 559567. Gottsberger, G., Silberbauer-Gottsberger, I., 1988. Evolution of ower structures and pollination in Neotropical Cassinae (Caesalpiniaceae) species. Phyton (Austria) 28, 293320. Harley, M.M., 1992. The potential value of pollen morphology as an additional taxonomic character in subtribe Ociminae (Ocimeae: Nepetoideae: Labiatae). In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, pp. 125138. Harley, R.M., 1971. An explosive pollination mechanism in Eriope crassipes, a Brazilian Labiate. Biol. J. Linn. Soc. 3, 159162. Harley, R.M., Atkins, S., Budantsev, A., Cantino, P.D., Conn, B., Grayer, R.J., Harley, M.M., De Kok, R., Krestovskaja, T., Morales, A., Paton, A.J., Ryding, O., Upson, T., in press. Labiatae. In: J.W. Kadereit (ed.), The Families and Genera of Vascular Plants, vi (Lamiales), Springer, Berlin. Harley, R.M., Paton, A.J., Ryding, O., 2003. New synonymy and taxonomic changes in the Labiatae. Kew Bull. 58, 485489. Hasegawa, M., Kishino, H., Yano, T., 1985. Dating the humanape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 21, 160174. Hedge, I.C., 1972. The pollination mechanism of Aeollanthus njassae. Notes Roy. Bot. Gard. Edinburgh 32, 4548. Hegnauer, R., 1989. Chemotaxonomie der Panzen, vol. 8. Birkh auser, Basel. Huck, R.B., 1992. Overview of pollination biology in the Lamiaceae. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, pp. 502507. Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: bayesian inference of phylogenetic trees. Bioinformatics 17, 754755. Jamzad, Z., Grayer, R.J., Kite, G.C., Simmonds, M.S.J., Ingrouille, M., 2003. Leaf surface avonoids in Iranian species of Nepeta L. (Lamiaceae) and some related genera. Biochem. Syst. Ecol. 31, 587600. Kaufmann, M., Wink, M., 1994. Molecular systematics of the Nepetoideae (Family Nepetoideae): phylogenetic implications from rbcL gene sequences. Z. Naturforsch. C 49, 635645. Lawrence, B.M., 1992. Chemical components of Labiatae oils and their exploitation. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, pp. 399436. Li, X.W., Hedge, I.C., 1994. Labiatae. In: Wu, Z.Y., Raven, P.H. (Eds.), Flora of China, 17. Missouri Botanical Garden. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identication of Flavonoids. Springer Verlag, Berlin.
A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299 Vestri Alvatenga, S.A., Gastmans, J.P., Rodrigues, G.Do.V., Moreno, P.R.H., Emerenciano, V.De.P., 2001. A computer-assisted approach for chemotaxonomic studies-diterpenes in Lamiaceae. Phytochemistry 56, 583595. Wagsta, S., Olmstead, R., Cantino, P.D., 1995. Parsimony analysis of cpDNA restriction site variation in Subfamily Nepetoideae (Labiatae). Am. J. Bot. 82, 886892.
299
Wagsta, S., Hickerson, L., Spangler, R., Reeves, P.A., Olmstead, R.G., 1998. Phylogeny in the Labiatae s.l., inferred from cp DNA sequences. Pl. Syst. Evol. 209, 265274. Wilcox, T.P., Zwickl, D.J., Heath, T.A., Hillis, D.M., 2002. Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support. Mol. Phylogenet. Evol. 25, 361371.

Phylogeny and Evolution of Basils and Allies (Ocimeae, Labiatae) Based On Three Plastid DNA Regions

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Phylogeny and Evolution of Basils and Allies (Ocimeae, Labiatae) Based On Three Plastid DNA Regions

Uploaded by

Copyright:

Available Formats

MOLECULAR PHYLOGENETICS AND EVOLUTION

Molecular Phylogenetics and Evolution 31 (2004) 277299 www.elsevier.com/locate/ympev

Received 8 April 2003; revised 1 August 2003

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

AJ505412 AJ505333 AJ505414 AJ505426 AJ505411 AJ505334 AJ505406

AJ505356 AJ505419 AJ505357 AJ505358 AJ505422

280 Table 1 (continued)

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

rps16 Genbank Accession AJ505361

AJ505362 AJ505363 AJ505364 AJ505365 AJ505366 AJ505367 AJ505368

AJ505369 AJ505370 AJ505371

282 Table 2 (continued)

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

RI 0.75 0.81 0.75 0.92 1.0 0.0 0.89 0.76

RC 0.19 0.33 0.25 0.46 1.0 0.0 0.44 0.33

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

Hyptidinae Isodon Ocimum Coleus Platostoma Syncolostemon

0.4 0.35 0.95

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277299

You might also like