1-s2.0-s1055790303003099-main

MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 31 (2004) 277–299

EVOLUTION

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

Madeline M. Harley,a Fiona Willis,a Monique S.J. Simmonds,b Martyn P. Powell,b

and Vincent Savolainenb

a The Herbarium, Royal Botanic Gardens, Kew, Richmond TW9 3AB, UKb Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond TW9 3AB, UK

c Charles E. Moss Herbarium, School of Animal, Plant, and Environmental Science, University of the Witwatersrand,

Private Bag 3, Wits 2050, Gauteng Province, South Africa

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, trnL–trnF 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: Lav-

andulinae, Hyptidinae, Ociminae, and Plectranthinae. Only Lavandulinae has clear non-molecular synapomorphies. Several cur-

rently 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 flavones in the leaf exudates differ in their distributions across the

phylogeny and this could contribute to differences in the recorded medicinal as well as pesticidal uses of the species in the different

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 five times, and New World colonisation occurred at least

three times.

� 2003 Elsevier Inc. All rights reserved.

1. Introduction

Basils and their allies, classified 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: [email protected] (A.J. Paton).1 Present address: Forest Herbarium, Royal Forest Department,

Bangkok 10900, Thailand.

1055-7903/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2003.08.002

et al., in press). The group is economically and medici-

nally 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

properties (Githinji and Kokwaro, 1993; Riviera-Nu~nezand Ob�on De Castro, 1992).

Several genera currently recognised within Ocimeae,

such as Plectranthus, have no clear synapomorphies and

could be polyphyletic. Although a few molecular phy-

logenic trees of Lamiaceae and subfamily Nepetoideae

have been produced using plastid DNA restriction site

mail to: [email protected]

278 A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277–299

data (Wagstaff et al., 1995), rbcL (Kaufmann and Wink,1994), rbcL and ndhF (Wagstaff et al., 1998), none deal

in any detail with Ocimeae. Even in the cases of Ocimum

and Platostoma where morphological parsimony anal-

yses have been published (Paton, 1997; Paton et al.,

1999), nodes lack statistical support. Robust phyloge-

netic analyses are much needed in order to efficiently

explore the medicinal and economic uses of the group.

The aims of this paper are first, to provide a phyloge-netic tree of tribe Ocimeae based on three plastid DNA

regions (trnL intron, trnL–trnF intergene spacer, and

rps16) and to evaluate the monophyly of the tribe; sec-

ond, 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 mono-

phyly of Ocimeae, the analysis also included a repre-sentative sample of 12 species from the remainder of

subfamily Nepetoiodeae, covering the subtribes Nepe-

teae, 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

Wagstaff et al. (1998).The list of plant samples with voucher information and

GenBank/EMBLAccession numbers is provided inTable

1. Total DNA was extracted from fresh, silica gel-dried,

and herbarium specimens using the 2� CTABmethod of

Doyle andDoyle (1987) and subsquently purified through

caesium chloride gradient. The trnL–trnF region (trnL

intron and trnL–trnF intergene) was amplified with

primers c (50-CGAAATCGGTAGACGCTACG-30) andf (50-ATTTGAACTGGTGACACGAG-30) from Taber-

let et al. (1991) using the following PCR program: 30 cy-

cles, 1min denaturation at 95 �C, 30 s annealing at 50 �C,1min extension at 72 �C, and 7min final extension. The

rps16 intron was amplified using primers rpsF (50-GT

GGTAGAAAGCAACGTGCGACTT-30) and rps2R

(50-TCGGGATCGAACATCAATTGCAAC-30; Oxel-

man et al., 1997). Amplification products were then pu-rified using QIAquick columns (Qiagen). DNA cycle

sequencing with BigDye terminators (PE Applied Bio-

systems) and PCR primers was performed in 5 ll volumes

on the cleaned PCRproducts (26 cycles, 10 s denaturation

at 96 �C, 5 s annealing at 50 �C, and 4min extension at

60 �C). Cycle sequencing reactions were purified by eth-

anol precipitation and run on an Applied Biosystems 377

automated DNA sequencer following the manufacturer�sprotocols (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 macro-

morphological characters were identified (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 34–38). 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 ex-

amined 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 morphol-

ogy will be discussed in detail in another paper, but

features that may represent synapomorphies of partic-

ular 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 char-

acters, bootstrap resampling, and Bayesian inference.

Maximum parsimony analysis was performed on the

trnL–F region and rps 16 intron combined and sepa-

rately and on the morphological data separately usingthe program PAUP*4.0b10 (Swofford, 2001). Most-

parsimonious trees with molecular and morphological

characters were obtained from 1000 replicates of ran-

dom taxon addition using equally weighted (EW) char-

acters (Fitch, 1971) and tree bisection–reconnection

(TBR) branch swapping with 10 trees held at each

step. These trees were also used to reweight the char-

acters according to the best fit of their rescaled consis-tency 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 effect, if any, of unstable taxa

(Farris, 1969).

Morphological data were also optimised on one of

the EW trees produced from the analysis of all DNAregions 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 morpho-

logical synapomorphies.

Bootstrap (BS) resampling (Felsenstein, 1985)

was performed on the trnL–F region and rps16 intron

Table 1

List of plant samples with voucher information and GenBank/EMBL Accession numbers

trnL–trnF Genbank

Accession

rps16 Genbank

Accession

Voucher

Aeollanthus buchnerianus Briq. AJ505434 AJ505327 Cult., K-1970-2734, Brummitt 10401, (K)

Aeollanthus densiflorus Ryding AJ505435 AJ505328 Cult., K-1970-3760, Mathew 6137 (K)

Alvesia rosmarinifolia Welw. AJ505436 AJ505329 Harder, Luwilka & Zimba 3634 (K)

Anisochilus harmandii Doan AJ505437 AJ505330 Suddee 775 (BKF, K, TCD)

Anisochilus pallidus Benth. AJ505438 AJ505331 Suddee et al.1080 (BKF, K, TCD)

Basilicum polystachyon (L.) Moench AJ505439 AJ505332 Greenway & Kanuri 14501 (K)

Callicarpa americana L. AJ505535 — Cult., K-0818400507 (K)

Callicarpa japonica Thunb. AJ505536 AJ505412 Cult., K-1934-12904 (K)

Capitanopsis angustifolia (Moldenke) Capuron AJ505440 AJ505333 Clement, Phillipson & Mantanantsoa 2117 (K)

Catoferia chiapsis Benth. AJ505537 AJ505414 Cult., K-1983-661, Chase 1149 (K)

Clinopodium vulgare L. subsp. arundanum AJ505547 AJ505426 Cult., K-453-79-04649 (K)

Congea tomentosa Roxb. AJ505530 AJ505411 Wagstaff, s.n. (BHO)

(Wagstaff and Olmstead, 1996)

Dauphinea brevilabra Hedge AJ505441 AJ505334 Cult., K-1998-2417, Hardy & Rauh 2876 (K)

Elsholtzia stauntonii Benth. AJ505526 AJ505406 Wagstaff 356, (BHO) (Wagstaff et al. 1995)

Endostemon obtusifolius (Benth.) N.E.Br. AJ505442 — Cult., K-1977-4733, Goyder & Paton 4105 (K)

Fuerstia africana T.C.E.Fr. AJ505550 AJ505427 G. Simon & M. Meliyo 228 (K)

Gmelina hystrix Kurz AJ505527 AJ505407 Cult., K-381-74-02999 (K)

Haumaniastrum katangense (S.Moore) Duvign. &

Plancke

AJ505540 AJ505417 Cult., K-1995-1197, Chase 13330 (K)

Hemizygia albiflora (N.E.Br.) Ashby AJ563764 AJ563783 Otieno 66 (J)

Hemizygia foliosa S.Moore AJ563765 AJ563784 Otieno 45 (J)

Hemizygia incana Codd AJ563766 AJ563785 Otieno 75 (J)

Hemizygia modesta Codd AJ563767 AJ563786 Otieno 120 (J)

Hemizygia obermeyerae Ashby AJ563768 AJ563787 Otieno 13 (J)

Hemizygia parvifolia Codd AJ563769 AJ563788 Otieno 71 (J)

Hemizygia persimilis (N.E.Br.) Ashby AJ563770 AJ563789 Otieno 76 (J)

Hemizygia pretoriae (G€urke) Ashby AJ563771 AJ563790 Otieno 50 (J)

Hemizygia punctata Codd AJ563772 AJ563791 Otieno 72 (J)

Hemizygia stalmansii A.J.Paton & K.Balkwill AJ563773 AJ563792 Otieno 111 (J)

Hemizygia subvelutina (G€urke) Ashby AJ563774 AJ563793 Otieno 69 (J)

Hemizygia transvaalensis (Schltr.) Ashby AJ563775 AJ563794 Otieno 56 (J)

Hemizygia teucriifolia (Hochst.) Briq. AJ563776 AJ563795 Otieno 64 (J)

Lavandula buchii Webb & Berthel. AJ505460 AJ505346 Upson 299 (RNG)

Lavandula maroccana Murb. AJ505461 AJ505347 Upson, s.n. (RNG)

Lavandula minutolii C. Bolle AJ505462 AJ505348 Upson s.n (RNG)

Lavandula rotundifolia Benth. AJ505463 AJ505349 Upson s.n (RNG)

Melissa officinalis L. AJ505529 AJ505410 Wagstaff 88-09 (BHO) (Wagstaff et al., 1995)

Mentha suaveolens Ledeb. AJ505541 AJ505418 Cult., K-1970-3169 (K)

Nepeta fissa C.A.Mey. AJ505430 AJ505323 Jamzad & Nikchehreh 80486 (TARI)

Nepeta menthoides Boiss. & Buhse AJ505431 AJ505324 Jamzad s.n. (K)

Nepeta racemosa Lam. AJ505432 AJ505325 Jamzad s.n. (TARI)

Nepeta straussii Hausskn. & Bornm. AJ505433 AJ505326 Jamzad et al. 76846 (TARI)

Ocimum americanum L. var. pilosum (Willd.)

A.J.Paton

AJ505464 AJ505350 Cult., Suddee 1145 (K)

Ocimum basilicum L. AJ505465 AJ505351 Cult., Suddee et al. 894 (BKF,K)

Ocimum citriodorum Vis. AJ505548 AJ505420 Chase 9939 (K)

Ocimum filamentosum Forssk. AJ505466 AJ505352 Brummitt 18993 (K)

Ocimum gratissimum L. var. gratissimum AJ505467 AJ505353 Cult., Suddee & Meade 1139 (BKF)

Ocimum gratissimum L. var. macrophyllum Briq. 1 AJ505468 AJ505354 Cult., K-1993-1030, Schellingerhaut s.n. (K)

Ocimum gratissimum L. var.macrophyllum Briq. 2 AJ505469 AJ505355 Cult., Suddee et al. 891/1 (BKF, K)

Ocimum labiatum (N.E.Br.) A.J.Paton1 AJ505470 — Edwards s.n. (K, NU)

Ocimum labiatum (N.E.Br.) A.J.Paton2 AJ505471 AJ505356 Balkwill et al. 10848 (K)

Ocimum selloi Benth. AJ505542 AJ505419 Gentry & Zardinini 49819 (K)

Ocimum serratum (Schltr.) A.J.Paton AJ505472 AJ505357 Balkwill et al. 10861 (J, K)

Ocimum tenuiflorum L. AJ505473 AJ505358 Cult., Suddee et al.893 (BKF, K, TCD)

Origanum vulgare L. AJ505543 AJ505422 Cult., K-000-69-19317, Chase 13334 (K)

Orthosiphon aristatus (Blume) Miq. AJ505474 — Suddee et al. 969 (BKF, K, TCD)

Orthosiphon parishii Prain AJ505475 AJ505359 Suddee & Puudjaa 1118 (BKF, K, TCD)

Orthosiphon rotundifolius Doan AJ505476 — Suddee et al. 944 (BKF, K, TCD)

Orthosiphon rubicundus (D.Don) Benth. AJ505477 AJ505360 Suddee 809 (BKF, TCD)

Platostoma africanum P.Beauv. AJ505478 — Pawek 4584 (K)

A.J. Paton et al. / Molecular Phylogenetics and Evolution 31 (2004) 277–299 279

Table 1 (continued)

trnL–trnF Genbank

Accession

rps16 Genbank

Accession

Voucher

Platostoma annamense (G.Tayl.)A.J.Paton AJ505479 AJ505361 Suddee et al.1028 (BKF)

Platostoma calcaratum (Hemsl.) A.J.Paton var.

garretii (Craib) S.Suddee, in ed.

AJ505480 — Suddee et al.910 (BKF, K, TCD)

Platostoma cambodgense (Doan) A.J.Paton var.

cambodgense

AJ505481 AJ505362 Chalermklin & Meade 98-10-16-01 (BKF)

Platostoma cambodgense (Doan) A.J.Paton

var.subulatum S.Suddee in ed.

AJ505482 AJ505363 Suddee 767 (BKF, K, TCD)

Platostoma cochinchinense (Lour.) A.J.Paton AJ505483 AJ505364 Wongprasert, Suddee & Puudjaa s.n

(BKF, K, TCD)

Platostoma coeruleum (R.E.Fr.) A.J.Paton AJ505484 AJ505365 Bidgood et al.3835 (K)

Platostoma coloratum (D.Don) A.J.Paton var.

coloratum

AJ505485 AJ505366 Suddee et al.958 (BKF, K, TCD)

Platostoma coloratum (D.Don) A.J.Paton var.

minutum S.Suddee in ed.

AJ505486 AJ505367 Suddee et al.915 (BKF, K, TCD)

Platostoma fimbriatum A.J.Paton AJ505487 AJ505368 Suddee 823 (BKF, K, TCD)

Platostoma hildebrandtii (Vatke) A.J.Paton AJ505488 — Bally & Smith B14742 (K)

Platostoma hispidum (L.) A.J.Paton AJ505489 — Beusekom et al. 3624 (K)

Platostoma intermedium A.J.Paton AJ505490 AJ505369 Suddee et al.947 (BKF, K, TCD)

Platostoma kerrii (Doan) A.J.Paton AJ505491 AJ505370 Suddee 769 (BKF, K, TCD)

Platostoma mekongense S.Suddee in ed. AJ505492 AJ505371 Chalermklin & Meade 98-10-16-02 (BKF, K)

Platostoma ocimoides (G.Tayl.) A.J.Paton AJ505493 — Suddee 788 (BKF, K, TCD)

Platostoma rotundifolium (Briq.) A.J.Paton AJ505494 AJ505372 Gilbert & Phillips 9239 (K)

Platostoma rubrum (Doan) A.J.Paton AJ505495 AJ505373 Suddee et al.998 (BKF, K, TCD)

Platostoma siamense (Murata) A.J.Paton AJ505496 AJ505374 Suddee & Pooma 849 (BKF, K, TCD)

Platostoma tectum A.J.Paton AJ505497 AJ505375 Suddee at al 1005 (BKF, K, TCD)

Plectranthus albicalyx S.Suddee in ed. AJ505498 AJ505376 Suddee et al.868 (BKF, K, TCD)

Plectranthus alboviolaceus G€urke AJ505531 AJ505377 Cult., K-1970-3926, Chase 9315 (K)

Plectranthus ambonicus (Lour.) Spreng. AJ505499 AJ505377 Suddee at al 869 (BKF)

Plectranthus barbatus Andr. AJ505500 AJ505378 Cult., K1982-5914, Thulin 4380 (K)

Plectranthus buchananii Bak. AJ505501 AJ505379 Cult., K-1970-3559, Brummitt 11597 (K)

Plectranthus calycinus Benth. AJ505502 AJ505380 Balkwill et al.10880 (J, K)

Plectranthus ciliatus E.Mey AJ505532 AJ505409 Cult., K-1991-6, Chase 13336 (K)

Plectranthus coeruleus (G€urke) Agnew AJ505503 AJ505381 Cult., K-1955-42604, Delap s.n. (K)

Plectranthus crassus N.E.Br. AJ505504 AJ505382 Cult., K-1970-2059, Brummitt 9700 (K)

Plectranthus cylindraceus Benth. AJ505538 AJ505383 Cult., K-1996-1453, Chase 8518 (K)

Plectranthus fredricii (G.Taylor) A.J.Paton, in ed. AJ505505 AJ505384 Cult., K-1999-15, RHS Wisley (K)

Plectranthus glabratus (Benth.) Alston AJ505508 AJ505387 Wongprasert, Suddee & Puudjaa s.n

(BKF, K, TCD)

Plectranthus helferi Hook. f. 1 AJ505509 AJ505388 Suddee & Puudjaa 1098 (BKF, K, TCD)

Plectranthus helferi Hook. f. 2 AJ505552 AJ505429 Chase 9768 (K)

Plectranthus laxiflorus Benth. AJ505510 AJ505389 Edwards s.n (K)

Plectranthus oertendahlii T.C.E. Fr. AJ505534 — Cult., K-1969-5789, Chase 3380 (K)

Plectranthus parishii Prain AJ505511 AJ505390 Suddee 11444 (BKF, K, TCD)

Plectranthus petiolaris Benth. AJ505512 AJ505391 Cult., K-1996-2729, U of Natal (K)

Plectranthus puberulentus J.K.Morton 1 AJ505506 AJ505385 K-1970-3763, Mathew 6158 (K)

Plectranthus puberulentus J.K.Morton 2 AJ505507 AJ505386 Cult., K-1970-3784, Mathew 6830 (K)

Plectranthus sanguineus Britten AJ505513 AJ505392 Cult., K-1970-2072, Brummitt s.n (K)

Plectranthus scutellaroides (L.) R.Br. AJ505514 AJ505393 Cult., Suddee et al.1094 (BKF, K, TCD)

Plectranthus thyrsoideus (Bak.) B.Matthew AJ505533 AJ505405 Cult., K-5638704012, Chase 13332 (K)

Plectranthus xerophilus Codd AJ505515 AJ505394 Cult., k-1989-1322, Hardy 6735 (K)

Prosthanthera nivea Benth. AJ505524 AJ505403 M.W. Chase 6980 (K)

Prosthanthera petrophila B.J.Conn AJ505525 AJ505404 M.W.Chase 6975 (K)

Puntia stenocaulis Hedge AJ505545 AJ505424 M T Thulin 10506 (UPS)

Pycnostachys reticulata (E.Mey.) Benth. AJ505516 AJ505395 Cult., K-1999-2425, Nat Bot. Gar. S. Africa (K)

Pycnostachys umbrosa (Vatke) Perkin AJ505517 AJ505396 Cult., k-1970-3755, Mathew 6067 (K)

Pycnostachys urticifolia Hook. AJ505518 AJ505397 Cult., K-1999-2426, Nat. bot. gar. s.Africa (K)

Rosmarinus officinalis L. AJ505546 AJ505425 Cult., K-1975-1177, Chase 13331 (K)

Salvia guaranitica Benth AJ505549 AJ505421 Cult., K-1973-14217 (K)

Syncolostemon argenteus N.E.Br AJ563777 AJ563796 Otieno 93 (J)

Syncolostemon comptonii Codd AJ563778 AJ563797 McCallum 916 (J)

Syncolostemon flabellifolius (S.Moore) A.J.Paton AJ563779 AJ563798 Linder 3981 (J)

Syncolostemon macranthus (G€urke) Ashby AJ563780 AJ563801 Otieno 109 (J)

Syncolostemon parviflorus Benth. AJ563781 AJ563800 Otieno 84 (J)


Table 1 (continued)

trnL–trnF Genbank

Accession

rps16 Genbank

Accession

Voucher

Syncolstemon rotundifolius Benth. AJ505523 AJ505402 Balkwill & Manning 414 (K)

Tectona grandis L. f. AJ505528 AJ505408 Waimea 73P172 (Wagstaff and Olmstead, 1996)

Tetradenia nervosa Codd AJ505520 AJ505399 Cult., K-1993-3116, Hardy 2910B (K)

Tetradenia fruticosa Benth. AJ505519 AJ505398 Cult., K-1989-1324, Hardy 2910A (K)

Thorncroftia longifolia N.E.Br. AJ505521 AJ505401 McDade LM 1281 (J)

Thorncroftia media Codd AJ505522 AJ505400 Cult., K-1993-3115, Hardy 3966 (K)

Thymus serpyllum L. var. citriodorum AJ505544 — Cult., K-1975-1177, Chase 13331 (K)

Vitex trifolia L. AJ505539 AJ505416 TCMK 15, Chase 8757 (K)

Table 2

Characters and coding used in the morphological MP analysis and mapping morphological and chemical characters and geographical distribution to

the phylogeny

CI RI RC

1. Fertile bracts: uniformly green (0); basally coloured (1) 0.33 0.89 0.3

2. Peduncle: absent (0); present (1) 0.15 0.65 0.1

3. Cyme branching: diachasial (0); cincinnate (1); inapplicable (2) 0.12 0.7 0.08

4. No. of flowers in cyme: 1 (0); 1–3 variable (1); 3 uniform (2); >3 (3) 0.25 0.55 0.16

5. Bracteoles: present (0); absent (1) 0.25 0.91 0.23

6. Cyme positioning: opposite (0); spiral (1); one-sided (2) 1.0 1.0 1.0

7. Cyme attachment: pedicellate (0); absent (1); sessile by pedicel fusion to calyx (2) 0.33 0.69 0.23

8. Pedicel attachment: median (0); opposite posterior lip (1) 0.5 0.95 0.48

9. Calyx circumscissile: not circumscissile (0); circumscissile (1) 0.5 0.5 0.25

10. 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)

0.28 0.7 0.20

11. Division of anterior calyx lobes: to same depth as lateral and anterior (0); fused—to less depth

than anterior/lateral split (1); split—to a greater depth than anterior/lateral split (2).

0.13 0.66 0.09

12. Shape of anterior calyx lobes: lanceolate (0); subulate (1); spinescent (2); sinuate (3); rounded

(4); inconspicuous (5)

0.26 0.74 0.19

13. Base of corolla tube: parallel sided (0); gibbous (1); saccate (2); spurred (3) 0.2 0.6 0.12

14. Lateral corolla lobe position: forming part of posterior lip (0); free—lateral (1); part of

anterior lip (2); absent (3)

0.18 0.76 0.14

15. Posterior/anterior corolla lip ratio: > or equal to 0.8 (0); <0.8 (1) 0.09 0.74 0.07

16. Tube shape: straight (0); curved down (1); sigmoid (2) 0.14 0.74 0.11

17. Anterior lobe hinged: absent (0); present (1) 1.0 1.0 1.0

18. Anterior lobe reflexed after anthesis: absent (0); present (1) 0.04 0.52 0.02

19. Stamen position: ascending (0); declinate (1); spreading porect (2) 0.4 0.76 0.34

20. Length of stamen: posterior exceeding anterior (0); anterior exceeding posterior (1); equal (2) 0.33 0.52 0.17

21. 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)

0.4 0.83 0.33

22. Anterior stamen fusion: absent, no fusion (0); present, fusion of the anterior pair of stamens

only, this pair separated from the posterior stamens (1)

1.0 1.0 1.0

23. Attachment of posterior stamen: Base to mid (0); mid to throat (1); throat—anterior side (2); throat—

regular separation (3)

0.38 0.86 0.32

24. Posterior filament form: tapering—no appendage (0); small tuft of hairs at base (1); basally swollen (2);

appendiculate—articulate (3); appendiculate—flattened (4); bent not appendiculate (5)

0.38 0.81 0.3

25. Anthers: with convergent thecae (0); synthecous (1); parallel (2) 0.67 0.94 0.63

26. Style: bifid (0); clavate (1); capitate—broad lobes (2); emarginate (3); bifid unequal (4) 0.27 0.53 0.14

27. Stylopodium: absent (0); present (1) 0.5 0.5 0.25

28. Style shield: absent (0); present (1) 0.33 0.0 0.0

29. Disk lobing: equally lobed (0); anterior larger (1), lobes opposite nutlets, not applicable (2) 0.13 0.84 0.11

30. Position of disk lobes: alternate with nutlets (0); opposite nutlets (1) 1.0 1.0 1.0

31. Nutlet areole: present (0); absent (1); not applicable, carpel not divided (2) 0.4 0.87 0.35

32. Fruit: divided into mericarps (0); undivided (1). 0.5 0.8 0.4

33. Anthers attachment: Thecae lateral (0); dorsifixed (1). 1.0 1.0 1.0

Pericarp anatomy characters

34. Number of cells in schlerenchyma region: >1 (0); 1 (1) 1.0 1.0 1.0

35. Shape of cells in schlerenchyma region: star- or stone-shaped (0); bone-shaped arranged

vertically (1)

1.0 1.0 1.0

36. Crystals in schlerenchyma region: absent (0); present (1) 0.56 0.91 0.51


Table 2 (continued)

CI RI RC

37. Plate-shaped content in exocarp cells: absent (0); present (1) 0.25 0.75 0.19

38. Postion of lumen in the bone cells: basal to central (0); apical (1); inapplicable (2) 0.4 0.81 0.33

Chemical characters mapped to phylogeny

39. 5-OH-6,7-diOMe-Flavones absent (0); present (1) 0.33 0.75 0.25

40. 5-OH-6,7,8-triOMe-Flavones absent (0); present (1) 0.5 0.92 0.46

41. 5,6-diOH-7,8-diOMe-Flavones absent (0); present (1) 1.0 1.0 1.0

42. Flavonols absent (0); present (1) 0.25 0.0 0.0

43. Quinonoid diterpenoids absent (0); present (1) 0.5 0.89 0.44

44. Geographic distribution Tropical Africa (0); Madagascar (1); SE Asia (2); Mediterranean/Central Asia

(3); Macaronesia (4); Australia (5); New World (6)

0.42 0.76 0.33

Statistics were calculated based on the characters fit to the topology of the equally weighted molecular tree shown in Fig. 3.


combined and on morphological characters separately,

using PAUP* 4.0b10 (Swofford, 2001). We performed in

each case 500 replicates with TBR swapping algorithm,

with EW and SW, and holding five 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 andRonquist, 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 ngen¼ 500,000,

printfreq¼ 100, samplefreq¼ 10, nchains¼ 4). Because

HKY85 (Hasegawa et al., 1985) is a good compromise

between the complexity of the model of DNA substi-

tution (transition/transversion ratio plus differentialnucleotide composition) and time requirement for its

computation, we set up this model as follows: lset

nst¼ 2 rates¼ equal. The first 15,000 trees were dis-

carded to allow for the ‘‘burn-in’’ of the process.

Biogeographic relationships were explored by opti-

mising the geographic distribution of terminal taxa

(Table 2, Appendix A) on a ultrametric tree in McClade

3.08 (Maddison and Maddison, 1999) with ACCTRANoptimisation and polytomies treated as hard polytomies.

The ultrametric tree was produced using the non-para-

metric rate smoothing (NPRS) method of Sanderson

(1997) implemented in TreeEdit (v1.0 alpha 4-61, writ-

ten by A. Rambaut and M. Charleston; http://www.

evolve.zps.ox.ac.uk/software/TreeEdit/main.html). Maxi-

mum parsimony EW branch lengths resulting from the

molecular analysis were optimised onto the trees beforeNPRS.

2.3. Chemical characters

Flavonoid and diterpene constituents were studied in

19 species of Plectranthus and related genera (Pycno-

stachys, Tetradenia, Thorncroftia, Aeollanthus, and

Dauphinea), one species each of Endostemon andBasilicum. Results are also included from five 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 ar-ray detection and atmospheric pressure chemical ioni-

sation mass spectrometry as described previously

(Grayer et al., 2001).

HPLC chromatograms were examined at different

wavelengths (270, 335, and 420 nm) suitable to detect

both exudate flavonoids and quinonoid diterpenoids,

and the presence or absence of these compounds was

scored. Exudate flavonoids were grouped on the basis oftheir UV spectra into flavones and flavonols (Mabry

et al., 1970) and identified according to the HPLC

method developed by Grayer et al. (2001) and by co-

chromatography with standards.

The flavones were further subdivided according to the

three main A-ring hydroxylation/methoxylation substi-

tution patterns found in these species, 5-hydroxy-6,

7-dimethoxyflavones (5-OH-6,7-diOMe), 5-hydroxy-6,7,8-trimethoxyflavones (5-OH-6,7,8-triOMe), and 5,6-di-

hydroxy-7,8-dimethoxyflavones (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 dit-erpenoids (maxima in both UV and visible light).

They were not identified further, nor subdivided into

different classes.

The distribution of flavonoids and quinonoid dit-

erpenoids are provided in Appendix A. Data were op-

timised on the molecular phylogeny following the same

method as for the morphological characters (see Table

2). The characters were scored as missing for taxa notstudied.

http://www.morphbank.ebc.uu.se/mrbayes/

http://www.morphbank.ebc.uu.se/mrbayes/

http://www.evolve.zps.ox.ac.uk/software/TreeEdit/main.html

http://www.evolve.zps.ox.ac.uk/software/TreeEdit/main.html


3. Results

3.1. MP analyses

After performing phylogenetic analyses of the trnL–

trnF and rps16 regions separately, no hard incongru-

ences were found (no contradictory clades supported by

bootstrap >60%; data not shown). The congruence be-

tween the two data sets supports the likelihood that theytrack 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. 1A–C. Only

bootstrap support (BS) greater than 50%, calculated

with EW are reported. The EW analysis produced 40trees with CI¼ 0.56 and RI¼ 0.76. The SW analysis

produced 656 trees with CI¼ 0.92 and RI¼ 0.95. The

major difference 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).

Fig. 1. (A–C) One most parsimonious tree after successive weighting of m

percentages (below branches, italicised, equal weights). Arrows indicate bra

arrows) and equal (black arrows) weights. (A) Basal lineages; (B) Ociminae;

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 treethere is less than BS 50% for this. The sistership of

Ociminae and Plectranthinae is supported, though Oc-

iminae has BS< 50% (Fig. 1A). The relative positions of

the Hyptidinae, Isodon, and Ociminae plus Plectranthi-

nae 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

olecular data showing branch length (above branches) and bootstrap

nches collapsing in the strict consensus trees with successive (unfilled

(C) Plectranthinae.

Fig. 1. (continued)


Platostoma plus Basilicum and Haumaniastrum; and

Endostemon plus Puntia is supported by the SW analysis

only.Within Plectranthinae a clade comprising Pycnosta-

chys, Holostylon, Anisochilus, and species of Plectran-

thus formerly placed in Coleus is supported (BS 97%)

(Fig. 1C). Pycnostachys forms a monophyletic group

within this Coleus clade (BS 96%). A clade which con-

tains 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 CI¼ 0.31 and RI¼ 0.85. There is little resolution in

the strict consensus tree (data not shown). The SW

morphological analysis produced 1960 trees with

CI¼ 0.31 and RI¼ 0.85. No hard incongruences were

found with the molecular MP trees (no contradictory

clades supported by bootstrap >60%; data not shown).

Fig. 1. (continued)


Analyses combining morphological and molecular data

again produced trees that showed no hard incongru-ences 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 threecombined 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) andIsodon 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 Nautochi-

lus (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 allseparated into two parts, whereas the Syncolostemon

clade is fragmented into three (Figs. 2A–C).

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 separatedby branches showing low posterior probabilities (see

Section 4).


The Bayesian analysis does not include gap charac-ters. To try and assess the influence of this on the rec-

ognition of clades, the distribution of gap characters was

analysed and the results of a sample of clades is pre-

sented 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, propor-tion of gap characters defining them, and a similar pro-

portion of informative gap characters within them. The

Bayesian analysis supports theOcimum 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 charac-

ters on to the phylogeny

Morphological and chemical characters, their states,

consistency index (CI), retention index (RI), and re-

Fig. 2. (A–C) Phylogenetic tree depicted from Bayesian inference and s

(B) Ociminae plus Plectranthinae p.p.; (C) Plectranthinae p.p.

scaled consistency index (RC) after ACCTRAN opti-misation 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 cy-

mes is a synapomorphy for Pycnostachys within the

Coleus clade, while cyme inserted on one side of the

inflorescence axis is a synapomorphy for Aeollanthus

within the Plectranthus clade (character 6, Table 2). Ahinged anterior lobe of the corolla is a synapomorphy

for Hyptidinae (character 17, Table 2). Anterior sta-

men fusion is synapomorphic for Syncolostemon

(character 22, Table 2). Disk lobes opposite the nutlets

is a synapomorphy for Lavandula (character 30, Table

2), and dorsifixed 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

howing posterior probabilities above branches. (A) Basal lineages;

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 synapo-

morphy for Ocimeae (character 25, CI¼ 0.66, Table 2).

The CI is less than one due to changes among other

states of this multistate character elsewhere in theanalysis.

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 taxono-mies gave support. Recently, Douady et al. (2003) and

Fig. 2. (continued)

Table 3

Table detailing support for selected clades

Clade Proportion of characters

which are gaps on

defining branch/branch

length in MP Analysis

Proportion of informative

characters within clade

which are gaps in MP

Analysis

Bayesian support

(posterior probability)

Bootstrap support

EW MP analysis

omitting gaps (%)

Bootstrap support

EW MP analysis,

gaps included (%)

Hyptidinae 0.4/17 0.5 0.4 95 89

Isodon 0.7/7 0.41 0.35 58 96

Ocimum 0.28/7 0.33 0.95 54 63

Coleus 0.28/7 0.30 — 96 97

Platostoma 0.75/4 0.39 — 56 —

Syncolostemon No gaps/4 0.41 — 97 100


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 difficulties in

the interpretation of sometimes strongly conflicting re-

sults’’ (Douady et al., 2003; p. 248). These studies foundthat the relation between Bayesian posterior probabili-

ties and bootstrap percentages were highly variable and

not ‘‘interchangeable’’ (Douady et al., 2003), although

when a threshold value was chosen for accepting in-

correct 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,

fitting well with the findings 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 explora-tions of the behaviour of Bayesian reconstructions be-

fore 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

Fig. 3. (A–C) 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 figure. Key to characters: fl1¼ 5-OH-6,7-diOMe-Flavones, absent ()), present (+); fl2¼ 5-OH-6,7,8-triOMe-Flavones absent

()), present (+); fl3¼ 5,6-diOH-7,8-diOMe-Flavones absent ()), present (+), equivocal (?); Q¼quinonoid diterpenoids absent ()), present (+);

D¼declinate stamens absent ()), present (+), stamens spreading (s); H¼hinge of anterior corolla lobe absent ()), present (+). F¼ fusion of all

stamens absent ()), present (+); A¼ fusion of anterior stamens absent ()), present (+); S¼ sigmoid corolla tube absent ()), present (+). C (in (C)

only)¼ anterior corolla lobe deflexed present (+), absent unless marked.


et al., 2002, p. 361), ‘‘Bayesian analysis can be exces-

sively liberal when concatenated gene sequences are

used’’ (Suzuki and Glazko, 2002, p. 16138) arguing for

increased confidence in the conservative boostrap.

The topology derived from the molecular EW MPanalysis 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

groupwith amorphological synapomorphy of dorsifixed,

Fig. 3. (continued)


synthecous anthers. Tribe Elsholtzieae is sister to theOcimeae. This is in agreement with plastid DNA re-

striction site studies of Wagstaff et al. (1998), but not the

rbcL study of Kaufmann and Wink (1994) where Oci-

mum is embedded within the tribe Mentheae, and is

sister to Melissa and Origanum [Majorana]. Subtribe

Lavandulinae, which can be diagnosed by the synapo-

morphy of having disk lobes opposite the nutlets, is

sister to the remainder of the Ocimeae. Examination of

pollen characters also suggests the presence of spinuloseelements on the aperture membrane as a putative syn-

apomorphy for Lavandulinae.

The relationships among Isodon, Hyptidinae, Plec-

tranthinae, and Ociminae are unresolved. The analysis

indicates that only the Hyptidinae has a clear non-mo-

lecular synapomorphy of a hinged anterior corolla lip.

However, Asterohyptis, not included in this analysis, but

normally placed within the Hyptidinae (Ryding, 1992)

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 mono-

phyletic. Within Ocimineae, Syncolostemon is mono-

phyletic 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 reticu-

lum 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 finedelicate net which, surprisingly, tends to survive ace-

tolysis intact, the lumina average upwards of 10–30 or

more in each primary lumen. These genera should

probably be merged under the earliest name Syncolos-

temon. This will be explored in more depth by one of us


(D.O.) in a separate paper. Although the relationshipsbetween 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 ofNautochilus, 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-mo-

lecular synapomorphies. It may be better to merge these

taxa under the earliest name Hoslundia Vahl. However,

as this will cause many changes in existing nomencla-

ture, we prefer to wait until further evidence for thisrelationship 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 inclu-

sion of Basilicum and Haumaniastrum. Further work is

needed to explore this relationship. A close relationship

between Puntia and Endostemon is also suggested by theSW 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 syn-

apomorphy for Pycnostachys, but Plectranthus longip-

etiolatus, not included in this analysis, also displays this

character. Cymes inserted on one side of the inflores-

cence axis is a synapomorphy for Aeollanthus. Aeollan-thus 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 Plectranthi-

nae. Some other clades have synapomorphies when

viewed only in the context of Plectranthinae. Tetradeniaand 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 molec-

ular phylogenetic analysis. There are two exceptions: (1)

Tetradenia which is similar in morphology to subtribeOciminae, 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 reducedflowers (ca. 3mm long) and lack morphological char-

acters 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 flower size.

4.2. Floral evolution and pollination

Ocimeae are usually considered to have stamens

pressed against the anterior, or lower side of the flower

(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 Ocimeaeinclude bees, butterflies, and flies (Huck, 1992; Nilsson

et al., 1985; Potgeiter et al., 1999; Van Der Pijl, 1972).

Declinate stamens are not a synapomorphy for the

Ocimeae (Figs. 3A–C): stamens are spreading in Pla-

tostoma caeruleum and in Tetradenia, the posterior pair

directed upwards and the anterior pair directed down-

wards. This change from declinate stamens has inde-

pendently occurred in these species. Apart from thischange, the position of the stamens relative to the co-

rolla 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 re-

supination of the whole corolla (Atkinson, 1998), rather

than a change in stamen position relative to the corollalobes.

There are several adaptations to pollinator behaviour

within Ocimeae. The anterior corolla lip of the corolla

can provide a landing platform. In Hyptidinae the sta-

mens 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


from the anthers (Harley, 1971). A similar explosivemechanism 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 platformthan 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 synapo-

morphic 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 lostseveral times within the Coleus clade, e.g., Plectranthus

cylindraceus and P. sanguineus (Fig. 3C).

Nilsson et al. (1985) suggest that a lackof afirm landing

platform in Plectranthus vestitus, which has a deflexed

anterior corolla lobe, favours hovering insects. Deflexed

anterior corolla lobes occur many times in Ocimeae

(CI¼ 0.04, Table 2; Fig. 3C, character only mapped

within Plectranthinae), and within the southern AfricanPlectranthinae this may be associated with long-tongued

hovering insects whose behaviour does not require a

landing platform (Nilsson et al., 1985). Deflexed lateral

corolla lobes seen in Thorncroftia may also be associated

with a reduction in landing platform. However, deflexed

anterior and free lateral corolla lobes are also found in

Lavandula, Endostemon, Puntia, and Tetradenia. In Lav-

andula, Tetradenia, and Platostoma caeruleum, the inflo-rescence is condensed. The close proximity of the corollas

and the spreading deflexed corolla lobes together present

a firm landing platform for insects, such as in bees visiting

Lavandula. In Endostemon and Puntia, however, the in-

florescence is not condensed.

Sigmoid corolla tubes are found in all the Coleus

clade and in Plectranthus laxiflorus/P. petiolaris subclade

of the Plectranthus clade (Fig. 3C). The sigmoid tube isalways associated with a horizontal anterior corolla lobe

(Fig. 3C). This combination of characters will favour

landing insects with flexible probosci. The need for in-

creased sampling and the lack of supported resolution at

the base of the Plectranthus clade, make it impossible to

draw firm conclusions about the direction of evolution

between straight tubes with deflexed lobes suiting hov-

ering pollinators, and sigmoid tubes with horizontalanterior lips suiting landing, flexible tongued insects.

Synthecous, dorsifixed 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 Holost-

ylon, a group of five species, the lower parts of the thecae

do not completely split forming a pouch and the upperparts 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 de-

pression at the apex, rather than bifid as in the re-mainder 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; Got-

tsberger and Silberbauer-Gottsberger, 1988). Gottsber-

ger and Silberbauer-Gottsberger (1988) suggest that

vibration of the stamens and dispersal of the pollen is

made more efficient by the rigidity of the stamens. Ri-gidity 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 pollinator�s body. The deeply

cucullate anterior corolla lobe of Holostylon may per-

form the same function. Buzz pollination has not been

recorded in the Lamiaceae and field work is needed toestablish if it does occur in Holostylon.

4.3. Phytochemistry

Overall, the quinone diterpenoids and various types

of flavonoids in the leaf exudates differ in their distri-

butions across the phylogeny (Fig. 3). For example, in

Plectranthinae there are no species with 8-oxygenatedflavones; they only contain 5-hydroxy-6,7-dimethoxyf-

lavones (Fig. 3C). In contrast, both 5-hydroxy-6,7-di-

methoxyflavones and 8-oxygenated flavones were

present in all species belonging to the Ocimum clade

(Fig. 3B). The profile of flavones of the genera Endos-

temon and Basilicum differed from Ocimum (Fig. 3B).

For example, Endostemon obtusifolius and Basilicum

polystachyon contain 5-hydroxy-6,7-methoxyflavonesbut no 8-oxygenated flavones. They also contain flavo-

nols which are absent in Ocimum.

Diterpenoids are produced by many species belong-

ing to most subfamilies of the Lamiaceae (Vestri Al-

vatenga 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 Oci-

mum, 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 flavones and lack quinonoid

diterpenoids (although they may produce different

types of diterpenoids), whereas most species of the

Coleus clade produce quinonoid diterpenoids and lack


flavonoids. Exceptions are P. cylindraceus, P. buchana-nii, and Holostylon katagense, which belong to the Co-

leus clade (Fig. 3C). The latter two species produce both

quinonoid diterpenoids and flavones, whereas P. cyl-

indraceus produces neither, but it does contain a wide

range of flavonols. These species with aberrent chemis-

try form a subclade within the Coleus clade in the SW

analysis (Fig. 1C) but not in the EW analysis (Fig. 3C).

These chemical differences within Ocimeae couldcontribute to differences in the recorded medicinal as

well as pesticidal uses of the species in the different

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 flavonoids 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 biologicalstudies. 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 po-

tential sister taxa within Viticoideae, the relative time of

the NPRS tree was not transformed into absolute agesas no clear calibration point could be identified. The

conclusions below should only be regarded as tentative

as only 12% of species are covered in the analysis.

Optimisation of geography on theNPRS 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 comprisingOciminae and Plectranthinae suggests an arrival in Africa

at a later relative time (Fig. 3A). Within this basally Af-

rican clade there are several secondary arrivals in Asia.

Such arrivals in the Platosoma, Orthosiphon, and in the

Plectranthus albicalyx/helfleri 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 andAsia 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. america-

num L. Several species ofOcimum and a few ofHyptis are

pan-tropical weeds.

Colonisation of Madagascar occurred at least twice in

the Plectranthinae clade (Fig. 3C): first in Capitanopsis/

Dauphinea and then in Tetradenia (Fig. 3C). However,these clades need to be more intensively sampled before

firm 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 Mada-

gascar, Tropical Africa, and Asia, but unfortunately se-quence data from Madagascan members of these clades

are not available. However, this suggests at least five ar-

rivals of Ocimeae in Madagascar in geological time.

Plectranthus luteus and P. tettensis and Pycnostachys

caerulea, all likely to fit into the Coleus clade, and End-

ostemon tenuiflorum are all found both in Tropical Africa

and Madagascar suggesting more recent movement be-

tween Madagascar and Africa.TribeOcimeae is mainly represented in theNewWorld

by subtribe Hyptidinae comprising around 380 species.

Ocimum has five 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 ispoor 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 gratissi-

mum, O. basilicum, and Plectranthus neochilus. These

species are widely used medicinally and horticulturallyand their distribution is heavily influenced 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 Maca-

ronesian regions. Tribe Ocimeae is represented in Aus-

tralia 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 isfound 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 Bio-

diversity 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


supporting Donald Otieno. We thank Richard Olmsteadfor sending DNA of Elsholtzia, Melissa, Tectona, and

Congea. We also thank Kevin Balkwill for collecting

material of Thorncroftia. We thank Olof Ryding for dis-cussion on scoring of pericarp characters and other

helpful comments.

Appendix A

Data matrix used for mapping morphological, chemical, and distribution characters onto phylogenetic trees. Char-acter order follows Table 2. A¼ (01) B¼ (12) C¼ (13) D¼ (23)

Aeollanthus buchnerianus
00201 20011 03100 10111 00201 00010 10101 11110 0000
Aeollanthus densiflorus
00201 20011 03000 10011 00201 00010 10101 11110 0100 Alvesia rosmarinifolia 002C1 00004 04200 10011 00201 00010 10101 000?? ???0
Anisochilus harmandii
00231 00100 00001 20011 00201 00010 10101 010?? ???2
Anisochilus pallidus
00231 00100 20001 20011 00201 00010 10101 010?? ???2
Basilicum polystachyon
00211 00000 01000 00011 00101 10000 10101 10?10 010E
Callicarpa americana
01030 00000 00010 00122 00002 00000 21010 102?? ???6
Callicarpa japonica
01030 00000 00010 00122 00002 00000 21010 102?? ???2
Capitanopsis angustifolia
01221 00000 00200 10111 00201 00010 101?? ??? ???1
Catoferia chiapensis
00231 02003 15010 00011 00101 30010 101?? ??? ???6 Clinopodium vulgare 00130 00001 01020 10101 00100 40000 000?? ??? 1??3
Congea tomentosa
01?30 00000 00010 00122 00102 00000 210AA 100?? ???2
Dauphinea brevilabra
00221 00003 10200 10111 00201 00010 101?? ???10 0001
Elsholtzia stauntonii
00130 00000 00010 00021 00100 00010 10001 000?? ???2
Endostemon obtusifolius
002C1 00000 00010 00111 00301 10100 10101 10?10 0100
Fuerstia africana
00201 00000 01000 00011 00101 10010 10101 10??? ???0
Gmelina hystrix
01010 00000 00010 00121 00002 40000 21010 102?? ???2
Haumaniastrum katangense
10221 00002 10100 00011 00021 00010 101?? ??? ???0 Hemizygia albiflora 00211 00000 01001 00011 01001 00010 10101 10??? ???0
Hemizygia foliosa
00201 00000 01001 00011 01001 00010 10101 10??? ???0
Hemizygia incana
00211 00000 01001 10111 01001 00010 10101 10??? ???0
Hemizygia modesta
00211 00000 01001 10111 01A01 00010 10101 10??? ???0
Hemizygia obermeyerae
00211 00000 01001 00011 01001 00010 10101 10??? ???0
Hemizygia parvifolia
00201 00000 01001 00011 01001 10010 10101 10??? ???0
Hemizygia persimilis
00211 00000 01001 00111 01A01 00010 10101 10??? ???0
Hemizygia pretoriae
00211 00000 01000 00111 01A01 00010 10101 10??? ???0 Hemizygia punctata 00201 00000 01000 00111 01001 00010 10101 10??? ???0
Hemizygia stalmansii
00201 00000 01001 00011 01001 00010 10101 10??? ???0
Hemizygia subvelutina
00211 00000 01001 00111 01001 00010 10101 10??? ???0
Hemizygia teucriifolia
00201 A0100 00000 20011 10201 30010 10101 01010 0010
Hemizygia transvaalensis
00211 00000 01001 00111 01001 00010 10101 10??? ???0
Hoslundia opposita
00201 00000 01000 00011 00101 10010 10101 10??? ???0
Hypenia brachystachys
01210 01001 00010 01111 00101 00010 00101 100?? ???6
Hypenia macrantha
01200 01000 00010 11111 00101 00010 00101 100?? ???6 Hypenia sp. 01200 01000 00010 11111 00101 00010 00101 100?? ???6
Hyptis brevipes1
01130 01000 01010 01111 00101 00000 00101 100?? ???6
Hyptis brevipes2
01130 01000 01010 01111 00101 00000 00101 100?? ???6
Hyptis capitata
01130 01000 01010 01111 00101 00000 00101 100?? ???6
Hyptis eriocephala
01130 01000 01010 01111 00101 00000 00101 100?? ???6
Hyptis floribunda
001C0 00000 00010 01111 00101 00000 00101 100?? ???6
Hyptis leptostachys ssp caatingense
00130 00000 01010 01111 00101 10000 00101 100?? ???6
Hyptis suaveolens
00130 00000 01010 01111 00101 00000 00101 100?? ???6 Isodon coetsa 01030 0000A 00100 10011 00001 00000 10101 000?? ???2
Isodon hispidus
01030 00001 00000 00011 00001 00000 10101 000?? ???2
Isodon lophanthoides
01030 00001 00000 00011 00001 00000 10101 000?? ???2
Isodon pharicus
01030 00000 00000 10011 00001 00000 10101 000?? ???3
Isodon rugosus
01030 00001 00100 00011 00001 00000 10101 000?? ???3


Appendix A (continued)

Isodon ternifolius
01030 00000 00100 10011 00001 00000 10101 000?? ???2
Lavandula buchii
00201 01001 00010 00111 00101 10001 10101 00011 0004
Lavandula maroccana
00201 01001 00010 00111 00101 10001 10101 00011 0003
Lavandula minutolii
00201 01001 00010 00111 00101 10001 10101 00011 0004
Lavandula rotundifolia
00201 01001 00010 00111 00101 10001 101?1 00011 0004 Mellissa officinalis 011C0 00001 01010 00001 00100 00100 100?? ??? ???3
Mentha suaveolens
001D0 00000 00010 00021 00100 00000 00001 100?? 1??3
Nepeta fissa
01130 00001 20010 10000 00300 00010 00001 A0011 1003
Nepeta menthoides
01130 00001 00011 10000 00300 00010 00001 A0011 1003
Nepeta racemosa
01130 00001 00011 10000 00300 00010 00001 A0011 1003
Nepeta straussii
01130 00001 00011 10000 00300 00010 00001 A0011 1003
Ocimum americanum var pilosum
00221 00000 00000 00011 00031 00000 10101 10?11 0002
Ocimum basilicum
00221 00000 00000 00011 00031 00000 10101 10?11 000D Ocimum citriodorum 00221 00000 00000 00011 00031 00000 10101 10?11 000D
Ocimum filamentosum
00221 00000 01000 00011 00031 00000 10101 10??? ???D
Ocimum gratissimum var gratissimum
00221 00000 10000 00011 00031 00000 101?? ???11 000D
Ocimum gratissimum varmacrophyllum1
00221 00000 10000 00011 00031 00000 101?? ??? ???D
Ocimum gratissimum varmacrophyllum2
00221 00000 10000 00011 00031 00000 101?? ??? ???D
Ocimum labiatum1
00211 00000 00000 10111 00001 00000 10101 101?? ???0
Ocimum labiatum2
00211 00000 00000 10111 00001 00000 10101 101?? ???0
Ocimum selloi
00221 00000 00000 00011 00031 00000 101?? ??? ???6 Ocimum serratum 00211 00000 00000 10111 00001 00000 10101 101?? ???0
Ocimum tenuiflorum
00231 00000 00000 00011 00001 00000 10101 10111 0002
Origanum vulgare
00200 00000 00000 00001 00100 00000 100?? ??? 1??3
Orthosiphon aristatus
002C1 00000 11000 00011 00101 10010 10101 100?? ???2
Orthosiphon parishii
00231 00000 01000 00011 00101 10010 10101 100?? ???2
Orthosiphon rotundifolius
00231 00000 11000 00011 00101 10010 10101 100?? ???2
Orthosiphon rubicundus
00211 00000 01000 00011 00101 10010 10101 100?? ???2
Platostoma africanum
10231 00001 15100 10011 00021 00010 10101 10??? ???0 Platostoma annamense 00231 00001 15300 10111 00021 00010 10101 10??? ???2
Platostoma calcaratum var garettii
10231 00001 15300 10111 00021 00010 10101 10??? ???2
Platostoma cambodgense var

cambodgense

10231
00001 15100 10011 00041 00010 10101 10??? ???2
Platostoma cambodgense var subulatum
10231 00001 15100 10011 00041 00010 10101 10??? ???2
Platostoma cochinchinense
10231 00001 15100 10011 00041 00010 10101 10??? ???2
Platostoma coeruleum
10131 00001 15100 10021 00021 00010 10101 10??? ???0
Platostoma coloratum var coloratum
10131 00001 10100 10011 00021 00010 10101 10??? ???2 Platostoma coloratum var minimum 10131 00001 10100 10011 00021 00010 10101 10??? ???2
Platostoma fimbriatum
10231 00001 15100 10011 00041 00010 10101 10??? ???2
Platostoma hildebrandtii
10231 00001 10100 10011 00021 00010 10101 10??? ???0
Platostoma hispidum
10231 00000 00000 10011 00021 00010 10101 10??? ???2
Platostoma intermedium
102C1 00001 15300 10011 00021 00010 10101 10??? ???2
Platostoma kerrii
10211 00001 15300 10011 00021 00010 10101 10??? ???2
Platostoma mekongense
10231 00001 15100 10011 00021 00010 10101 10??? ???2
Platostoma ocimoides
102C1 00001 15300 10011 00021 00010 10101 10??? ???2 Platostoma rotundifolium 10131 00001 15100 10011 00021 00010 10101 10??? ???0
Platostoma rubrum
10131 00001 15000 00011 00041 00010 10101 10??? ???2
Platostoma siamense
10131 00001 10100 10011 00021 00010 10101 10??? ???0
Platostoma tectum
10131 00001 15100 10011 00041 00010 10101 10??? ???2
Plectranthus albicalyx
00221 00100 10001 20011 10201 00010 10101 0?0?? ???2
Plectranthus alboviolaceus
00211 00003 01200 10011 20201 00010 10101 0?010 0000
Plectranthus amboinicus
00131 00100 00001 20011 10201 00010 10101 010?? ???0
Plectranthus barbatus1
00131 00100 00001 20011 10201 00010 10101 01000 0010 Plectranthus barbatus2 00131 00100 00001 20011 10201 00010 10101 0?000 0010


Appendix A (continued)

Plectranthus buchananii
00131 00100 00001 20011 10201 00010 10101 0?010 0010
Plectranthus calycinus
01131 00100 00200 20011 00201 00010 10101 0?0?? ???0
Plectranthus ciliatus
00211 00003 10200 00111 00201 00010 101?? ???10 0000
Plectranthus coeruleus
00221 00010 00001 B0011 10201 00010 10101 0?000 001A
Plectranthus crassus
00131 00100 20001 20011 10201 00010 10101 0?000 0010 Plectranthus cylindraceus 00131 00100 20001 20011 00201 00010 10101 01000 0100
Plectranthus fredricii
01131 00100 00001 20011 10201 00010 10101 00000 0010
Plectranthus helfleri1
00211 00100 00001 20011 10201 00010 10101 0?0?? ???2
Plectranthus helfleri2
00211 00100 00001 20011 10201 00010 10101 0?0?? ???2
Plectranthus glabratus
00131 00100 00001 20011 00201 00010 10101 0?0?? ???2
Plectranthus laxiflorus
01221 00000 00000 20011 00201 00010 10101 000?? ???0
Plectranthus oertendahlii
00211 00003 10200 10011 00201 00010 10101 01010 0000
Plectranthus parishii
01131 00100 00001 20011 00201 00010 10101 0?0?? ???2 Plectranthus petiolaris 01221 00000 00000 20011 00201 00010 10101 0?010 0000
Plectranthus puberulentus2
00131 00100 00001 20011 10201 00010 10101 0?0?? ???0
Plectranthus puberulentus1
00131 00100 00001 20011 10201 00010 10101 0?0?? ???0
Plectranthus sanguineus
00131 00100 20001 20011 00201 00010 10101 0?000 0010
Plectranthus scutellaroides
0A131 00100 10001 20011 10201 00010 10101 0?0?? ???2
Plectranthus thyrsoideus
00131 00100 00001 20011 10201 00010 10101 0???? ???0
Plectranthus xerophilus
00131 00100 00001 20011 10201 00010 10101 01000 0010
Prostanthera nivea
01200 00002 15120 00021 00000 02000 00010 102?? ???5 Prostanthera petrophila 01200 00002 15120 00021 00000 02000 00010 102?? ???5
Puntia stenocaulis
00201 00000 00010 00011 00301 00100 101?? ??? ???0
Pycnostachys reticulata
00201 11?00 02001 20011 10201 00010 10101 010?? ???0
Pycnostachys umbrosa
00201 11?00 02001 20011 10201 00010 10101 01000 0010
Pycnostachys urticifolia
00201 11?00 02001 20011 10201 00010 101?1 010?? ???0
Rosmarinus officinalis
00201 00002 00120 00102 00??? 40000 000?? ??? ???3
Salvia guaranitica
00131 00001 00020 20102 00??? 40010 10001 000?? ???6
Syncolostemon argenteus
00131 00001 00000 00111 01001 00010 10101 101?? ???0 Syncolostemon comptonii 00201 00000 01001 10111 01001 00010 10101 101?? ???0
Syncolostemon flabellifolius
00201 00000 00000 00011 01001 00010 10101 101?? ???0
Syncolostemon macranthus
00201 00000 01000 10111 01001 00010 10101 101?? ???0
Syncolostemon parviflorus
00201 00000 01000 00011 01001 00010 10101 101?? ???0
Syncolostemon rotundifolius
00201 00000 01000 00111 01001 00010 10101 101?? ???0
Tectona grandis
01030 00000 00010 00122 00102 02000 210?? ??? ???2
Tetradenia fruticosa
00211 00000 20010 00121 00101 00010 10101 000?? ???1
Tetradenia nervosa
00211 00000 20010 00121 00101 00010 10101 00010 0001 Thorncroftia longifolia 00201 00000 00010 00111 00201 00010 10101 000?? ???0
Thorncroftia media
00201 00000 00010 00111 00201 00010 10101 00010 0000
Thymus serphyllum var citriodorum
00130 00001 00020 00001 00100 00000 100?? ???11 1003
Vitex trifolium
010C0 00000 00010 00021 00002 00000 21010 102?? ???2
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monophyletic clades

allies lamiaceae

genera and1060 species

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annuallyfrom species

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subfamily nepetoideae