Palynological evolutionary trends within the tribe Mentheae with special emphasis on subtribe Menthinae (Nepetoideae: Lamiaceae)

Abstract

The pollen morphology of subtribe Menthinae sensu Harley et al. [In: The families and genera of vascular plants VII. Flowering
plants·dicotyledons: Lamiales (except Acanthaceae including Avicenniaceae). Springer, Berlin, pp 167–275, 2004] and two genera of uncertain subtribal affinities (Heterolamium and Melissa) are documented in order to complete our palynological overview of the tribe Mentheae. Menthinae pollen is small to medium
in size (13–43μm), oblate to prolate in shape and mostly hexacolpate (sometimes pentacolpate). Perforate, microreticulate
or bireticulate exine ornamentation types were observed. The exine ornamentation of Menthinae is systematically highly informative
particularly at generic level. The exine stratification in all taxa studied is characterized by unbranched columellae. Orbicules
are consistently absent in Menthinae. Our palynological data are interpreted in a phylogenetic context at tribal level in
order to assess the systematic value of pollen characters and to evaluate the existing molecular phylogenies for this group.
Pollen morphology suggests Heterolamium as a close relative of subtribe Nepetinae and supports the molecular affinity of Melissa to subtribe Salviinae.

Full text

ORIGINAL ARTICLE
Palynological evolutionary trends within the tribe Mentheae
with special emphasis on subtribe Menthinae
(Nepetoideae: Lamiaceae)
Hye-Kyoung Moon Æ Stefan Vinckier Æ
Erik Smets Æ Suzy Huysmans
Received: 13 December 2007 / Accepted: 28 March 2008 / Published online: 10 September 2008
Ó Springer-Verlag 2008
Abstract The pollen morphology of subtribe Menthinae
sensu Harley et al. [In: The families and genera of vascular
plants VII. Flowering plantsdicotyledons: Lamiales (except
Acanthaceae including Avicenniaceae). Springer, Berlin,
pp 167–275, 2004] and two genera of uncertain subtribal
affinities (Heterolamium and Melissa) are documented in
order to complete our palynological overview of the tribe
Mentheae. Menthinae pollen is small to medium in size
(13–43 lm), oblate to prolate in shape and mostly hexacol-
pate (sometimes pentacolpate). Perforate, microreticulate or
bireticulate exine ornamentation types were observed. The
exine ornamentation of Menthinae is systematically highly
informative particularly at generic level. The exine stratifi-
cation in all taxa studied is characterized by unbranched
columellae. Orbicules are consistently absent in Menthinae.
Our palynological data are interpreted in a phylogenetic
context at tribal level in order to assess the systematic value
of pollen characters and to evaluate the existing molecular
phylogenies for this group. Pollen morphology suggests
Heterolamium as a close relative of subtribe Nepetinae
and supports the molecular affinity of Melissa to subtribe
Salviinae.
Keywords Bireticulum  Mentheae  Menthinae 
Nepetoideae  Palynology  Phylogeny 
Exine ornamentation
Introduction
The pollen morphology of Lamiaceae has proven to be
systematically valuable since Erdtman (1945) used the
number of nuclei and the aperture number to divide the
family into two subfamilies (i.e. Lamioideae: bi-nucleate
and tricolpate pollen, Nepetoideae: tri-nucleate and hexa-
colpate pollen). While the circumscription of subfamily
Lamioideae has been dramatically changed after Erdtman’s
treatment due to progress in molecular systematics,
including for instance many genera previously placed in
Verbenaceae, the subfamily Nepetoideae is a consistently
supported monophyletic group based on both molecular
and morphological evidence (Cantino 1992; Harley et al.
2004; Wagstaff et al. 1995). Examples of morphological
synapomorphies for the latter subfamily are the presence of
six colpi, although some variation in aperture number
occurs sporadically, exalbuminous seeds and an investing
embryo type (Cantino 1992). According to the most recent
classification (Harley et al. 2004), Nepetoideae comprise
three tribes: Elsholtzeae, Mentheae and Ocimeae.
Palynological studies in Nepetoideae have been res-
tricted geographically or have been based on a limited
number of taxa using mainly light microscopic observa-
tions (Afzal-Rafii 1983; Henderson et al. 1968; Varghese
and Verma 1968; Vij and Kashyap 1975; Waterman 1960).
In contrast to the subfamily Lamioideae in which many
palynological data are published (Abu-Asab and Cantino
1992, 1993, 1994), only few genera of Nepetoideae are
described using detailed scanning and transmission electron
H.-K. Moon (&)  S. Vinckier  E. Smets  S. Huysmans
Laboratory of Plant Systematics, Institute of Botany
and Microbiology, K.U.Leuven, Kasteelpark Arenberg 31,
P.O.Box 2437, 3001 Louvain, Belgium
e-mail: hyekyoung.moon@bio.kuleuven.be
S. Vinckier
Center for Transgene Technology and Gene Therapy, Flanders
Institute for Biotechnology (VIB-3), K.U.Leuven,
Campus Gasthuisberg, Herestraat 49, 3000 Louvain, Belgium
E. Smets
National Herbarium of the Netherlands, Leiden University
Branch, P.O.Box 9514, 2300 RA Leiden, The Netherlands
123
Plant Syst Evol (2008) 275:93–108
DOI 10.1007/s00606-008-0042-y

microscopic observations (Harley 1992; Harley et al. 1992;
Rudall 1980; Wagstaff 1992).
Tribe Mentheae includes about 65 genera and 2,000
species, and represents almost one fourth of the currently
recognized genera in Lamiaceae. Mentheae is the largest
and economically most important tribe including many
culinary herbs such as mint (Mentha), oregano (Origanum),
savory (Satureja), sage (Salvia) and thyme (Thymus).
Mentheae, which was paraphyletic or polyphyletic as cir-
cumscribed by Briquet (1897) and Wunderlich (1967), was
greatly expanded by Cantino et al. (1992) to include
Glechoneae, Hormineae, Lepechinieae, Meriandreae,
Monardeae, Nepeteae, Prunelleae, Rosmarineae and Salv-
ieae and is strongly supported as a monophyletic group by
molecular data (Wagstaff et al. 1995, Table 1). More
recently, traditionally segregated tribes were recognized at
subtribal level by Harley et al. (2004), i.e., Salviinae,
Menthinae and Nepetinae. Although tribe Mentheae is a
monophyletic group, the monophyly of its subtribes is still
questionable based on the existing molecular phylogenies
(Trusty et al. 2004; Walker and Sytsma 2007).
Since a thorough palynological study of the entire tribe
Mentheae was lacking (Wagstaff 1992), we have presented
a detailed pollen study of subtribe Salviinae and Nepetinae
with a strong emphasis on the type genera Salvia and Ne-
peta (Moon et al. 2008a, b). In addition, we have assessed
the possible occurrence of orbicules in Salviinae and Ne-
petinae. Orbicules are small sporopollenin particles that can
be produced in species with a secretory tapetum (Huysmans
et al. 1998, 2000). The presence or absence of orbicules and
their morphology can provide additional palynological
characters that might have a phylogenetic significance (e.g.,
Huysmans et al. 1998; Raj and El-Ghazaly 1987; Vinckier
and Smets 2002). All taxa studied in Salviinae and Nepet-
inae have smooth tapetal membranes without any patterned
sporopollenin deposition, meaning that orbicules were not
observed in our two previous studies.
In order to present a detailed palynological overview of
the entire tribe Mentheae sensu Harley et al. (2004), the
present study focuses on the third subtribe Menthinae
including two unplaced genera Heterolamium and Melissa.
We, therefore, aim to document and illustrate the pollen
morphology as well as the ultrastructure of the pollen wall,
and we want to trace the possible occurrence of orbicules in
subtribe Menthinae sensu Harley et al. (2004) using LM,
SEM and TEM. These observations are discussed in the
light of our previous pollen studies in the two other sub-
tribes Salviinae and Nepetinae, with special emphasis on
the systematic significance of pollen characters within the
entire tribe Mentheae. Additionally, palynological data of
two currently unplaced genera Heterolamium and Melissa
are incorporated to comment on their enigmatic taxonomic
position.
Materials and Methods
Material
The present study is based on herbarium material of 58
species (66 specimens) from 42 genera of the subtribe
Menthinae, collected from the following herbaria: BR, GH,
K, LV, MO and S (acronyms follow Holmgren et al. 1990;
for a complete list of specimens, see Appendix) and also in
part from plants recently collected by the first author. Two
monotypic genera Eriothymus and Kurzamra could not be
studied because of lack of material. Since the genus Lyc-
opus has already been studied by Moon and Hong (2003), it
was also excluded from the present study.
SEM observations
Standard acetolysis (Erdtman 1960; Reitsma 1969)
destroys the colpus membranes, which may influences the
natural pollen shape and size (Demissew and Harley 1992;
Moon et al. 2008a, b). The subtribes Salviinae and Ne-
petinae showed a striking size difference according to
treatment: critical point dried pollen grains were always
smaller than acetolysed pollen grains (Harley 1992; Lens
et al. 2005; Moon et al. 2008a, b; Schols et al. 2004).
Taking this in consideration, all samples were critical point
dried in this study for optimally preserving their natural
size and shape.
For pollen and orbicule observations, dried flowers or
buds were rehydrated for 1–2 h in the wetting agent
Agepon
Ò
(Agfa Gevaert, Leverkusen, Germany; Agepon
wetting agent:distilled water = 1:200). Anthers were sep-
arated from the flowers and the tips were removed with a
razor blade to facilitate rehydration. After dissection, the
anthers remained for one more hour in the wetting agent.
Following dehydration in a graded acetone series, the
material was critical point dried (CPD 030, Balzers). The
dried anthers were mounted on stubs with double-sided
adhesive tape. The locules were opened and the pollen
grains were carefully removed with a cactus needle. The
removed pollen grains were collected on the same stub for
observation. The stubs were coated with gold (SPI-MOD-
ULE
TM
Sputter Coater, SPI Supplies, West Chester, PA,
USA) prior to observation with a JEOL JSM-6360 scanning
electron microscope at 10–20 kV. The size measurements
of 15–20 fully developed pollen grains on SEM images
were made using Carnoy 2.0 (Schols et al. 2002).
Ultrastructure
For transmission electron microscopy (TEM), the anthers
from living material were directly fixed in 2.5% glutaral-
dehyde while dried anthers were rehydrated in 0.05 M
94 H.-K. Moon et al.
123

Table 1 Genera of tribe Mentheae sensu Harley et al. (2004) and their previous taxonomic positions
Tribe Mentheae sensu Harley
et al. (2004)
Bentham (1876) Briquet (1897) Wunderlich (1967)
Subtribe Menthinae
Acanthomintha (A.Gray)
Benth. & Hook. f.
Tribe Satureineae, subtribe
Melisseae
Tribe Glechoneae Tribe Glechoneae
Blephilia Raf. Tribe Monardeae Tribe Monardeae Tribe Monardeae
Bystropogon L’He
´
r. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Cleonia L. Tribe Stachydeae Tribe Stachydeae, subtribe
Brunellinae
Tribe Stachydeae, subtribe
Prunellinae
Clinopodium L. Tribe Satureineae, subtribe
Melisseae (as Calamintha)
Genus Satureia sect. Clinopodium Genus Satureja
Conradina A. Gray Tribe Satureineae, subtribe
Melisseae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Cuminia Colla Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Menthinae
Tribe Saturejeae, subtribe
Menthinae
Cunila D. Royen ex L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Cyclotrichium (Boiss.)
Manden. & Scheng.
Genus Calamintha (see
Clinopodium)
Genus Satureja sect. Cyclotrichum N.I.
Dicerandra Benth. Tribe Satureineae, subtribe
Melisseae (as Ceranthera)
Tribe Satureieae, subtribe
Melissinae (as Ceranthera)
Tribe Saturejeae, subtribe
Melissinae (as Ceranthera)
Eriothymus (Benty.) Schmidt Tribe Satureineae, subtribe
Melisseae (as Keithia
)
Genus Hedeoma N.I.
Glechon Spreng. Tribe Satureineae, subtribe
Melisseae
Tribe Glechoneae Tribe Glechoneae
Gontscharovia Boriss. N.I. N.I. N.I.
Hedeoma Pers. Tribe Satureineae, subtribe
Melisseae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Hesperozygis Epling N.I. N.I. N.I.
Hoehnea Epling N.I. N.I. N.I.
Horminum L. Tribe Satureineae, subtribe
Lepechineae
Tribe Hormineae Tribe Hormineae
Hyssopus L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Hyssopinae
Tribe Saturejeae, subtribe
Hyssopinae
Kurzamra Kuntze Tribe Satureineae, subtribe
Melisseae (as Soliera)
Tribe Satureieae, subtribe
Melissinae
N.I.
Lycopus L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Menthinae
Tribe Saturejeae, subtribe
Menthinae
Mentha L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Menthinae
Tribe Saturejeae, subtribe
Menthinae
Micromeria Benth. Tribe Satureineae, subtribe
Melisseae
Genus Satureia Genus Satureja
Minthostachys (Benth.) Spach Genus Bystropogon Genus Bystropogon N.I.
Monarda L. Tribe Monardeae Tribe Monardeae Tribe Monardeae
Monardella Benth. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Neoeplingia Ramamoorthy,
Hiriart & Medrano
N.I. N.I. N.I.
Obtegomeria P.D. Cantino &
Doroszenko
N.I. N.I. N.I.
Origanum L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Pentapleura Hand.-Mazz. N.I. N.I. N.I.
Palynological evolutionary trends within the tribe Mentheae 95
123

Table 1 continued
Tribe Mentheae sensu Harley
et al. (2004)
Bentham (1876) Briquet (1897) Wunderlich (1967)
Piloblephis Raf. Genus Satureja (sect. Pycnothymus) Genus Satureja (sect.
Pycnothymus)
N.I.
Pogogyne Benth. Tribe Satureineae, subtribe
Melisseae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Poliomintha A. Gray Tribe Satureineae, subtribe
Melisseae
Genus Hedeoma N.I.
Prunella L. Tribe Stachydeae (as Brunella) Tribe Stachydeae, subtribe
Brunellinae (as Brunella)
Tribe Stachydeae, subtribe
Prunellinae
Pycnanthemum Michx. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae (as Koellia)
Tribe Saturejeae, subtribe
Thyminae
Rhabdocaulon (Benth.) Epling Tribe Satureineae, subtribe
Melisseae (as Keithia)
Genus Hedeoma N.I.
Rhododon Epling N.I. N.I. N.I.
Saccocalyx Coss. & Durieu Genus Satureja Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Satureja L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Stachydeoma Small Genus Hedeoma Genus Hedeoma N.I.
Thymbra L. Tribe Satureineae, subtribe
Melisseae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Thymus L. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Zataria Boiss. Tribe Satureineae, subtribe
Menthoideae
Tribe Satureieae, subtribe
Thyminae
Tribe Saturejeae, subtribe
Thyminae
Ziziphora L. Tribe Monardeae Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
Subtribe Salviinae
Chaunostoma Donn. Sm. N.I. Incertae Sedis N.I.
Dorystaechas Boiss. & Heldr. Tribe Monardeae Tribe Meriandreae Tribe Meriandreae
Lepechinia Willd. Tribe Satureineae, subtribe
Lepechineae
Tribe Lepechinieae Tribe Lepechinieae
Meriandra Benth. Tribe Monardeae Tribe Meriandreae Tribe Meriandreae
Perovskia Kar. Tribe Monardeae Tribe Meriandreae Tribe Meriandreae
Rosmarinus L. Tribe Monardeae Tribe Rosmarineae Tribe Rosmarineae
Salvia L. Tribe Monardeae Tribe Salvieae Tribe Salvieae
Zhumeria Rech.f. & Wendelbo N.I. N.I. N.I.
Subtribe Nepetinae
Agastache J.Clayton ex
Gronov.
N.I. Tribe Nepeteae Tribe Nepeteae
Cedronella Moench Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Dracocephalum L. Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Drepanocaryum Pojark. N.I. N.I. N.I.
Glechoma L. Genus Nepeta Tribe Nepeteae Tribe Nepeteae
Hymenocrater Fisch. &
C.A.Mey.
Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Lallemantia Fisch. &
C.A.Mey.
Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Lophanthus Adans. Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Marmoritis Benth. Genus Nepeta N.I. N.I.
Meehania Britton N.I. Tribe Nepeteae Tribe Nepeteae
96 H.-K. Moon et al.
123

sodium cacodylate buffer (pH 7.3) prior to fixation. Fixed
anthers were rinsed with 0.05 M sodium cacodylate buffer
(pH 7.3), followed by post fixation in 2% OsO
4
. Prior
to embedding in LR-White Resin (Polysciences Inc.,
Warrington, PA, USA), the material was dehydrated in a
graded ethanol series. Semi-thin sections (±1 lm) were cut
with a microtome (Reichert Jung Ultracut E), stained with
0.1% thionin–0.1% methylene blue, and observed with a
Leitz Dialux 20 microscope. The ultra-thin sections
(70 nm) on copper grids were stained with uranyl acetate
and lead citrate in an ultrastainer (LKB 2168), and
observed with a Zeiss EM 900 transmission electron
microscope at 50 kV.
Pollen terminology follows the Glossary of Pollen and
Spore Terminology (Punt et al. 2007; http://www.bio.uu.nl/
*palaeo/glossary/glos-int.htm).
Results
The pollen morphology of subtribe Menthinae is highly
uniform. Therefore, we prefer to give a general pollen
description for the subtribe with references to particular
species when necessary. The major pollen features of all
taxa investigated are summarized in Table 2. Representa-
tive pollen grains are illustrated in Figs. 1–4.
Size and shape
Pollen grains are shed as monads and their size varies from
small to medium (P = 13.0–43.3 lm). Mentha pulegium
possesses the smallest pollen grains while Cleonia lusita-
nica and Monarda punctata have the largest pollen grains
(Table 2). At infrageneric level the taxa studied have a
rather narrow size range (Table 2).
The shape of pollen grains in equatorial view ranges
from oblate to prolate (P/E = 0.67–1.72; Fig. 1a–f). Sub-
oblate to prolate-spheroidal shapes are common even
within the same taxa (Table 2). The outline in polar view is
circular, sometimes mixed with elliptic grains because of
width of the mesocolpium area (Fig. 1g–l).
Apertures
All taxa studied have hexacolpate pollen grains. Pollen
aperture heteromorphism only occurs in Prunella vulgaris
and Pycnanthemum incanum with a few pentacolpate pol-
len grains (Fig. 1j).
Simple colpi are distributed symmetrically. The range of
colpi lengths is 10.6–37.1 lm. Colpus length is strongly
correlated with length of polar axis. Colpi ends are acute
(Fig. 1) and the apocolpium index (AI) varies between 0.10
and 0.29 (Table 2).
Exine ornamentation
Three distinct exine ornamentation types are observed in
Menthinae: bireticulate, microreticulate or perforate (Fig. 2).
Based on the detailed configuration of the exine ornamen-
tation three subtypes can be defined for the bireticulate
pattern, and two subtypes for the microreticulate pattern.
Type I: bireticulate
A bireticulum consists of a non-congruent, two-layered
reticulum. The main reticulum is referred to as the primary
reticulum and the substratum as the secondary reticulum.
Three subtypes can be defined based on the number of
secondary lumina in each primary lumen. Type I-1: The
primary muri are more than twice as thick as the secondary
muri. Primary lumina are continuous and irregular to
rounded and slightly shallow. The secondary reticulum is
microreticulate and the number of secondary lumina does
not exceed ten in each primary lumen. Observed in: Gon-
tscharovia, Horminum, Melissa, Monardella, Thymus,
Ziziphora (Fig. 2a, b). Type I-2: The primary muri are
thicker than the secondary muri. The shape of the primary
lumina is rounded and continuous. The secondary lumina
Table 1 continued
Tribe Mentheae sensu Harley
et al. (2004)
Bentham (1876) Briquet (1897) Wunderlich (1967)
Nepeta L. Tribe Nepeteae Tribe Nepeteae Tribe Nepeteae
Schizonepeta (Benth.) Briq. Genus Nepeta Tribe Nepeteae Tribe Nepeteae
Incertae Sedis
Heterolamium C. Y. Wu N.I. N.I. N.I.
Melissa L. Tribe Satureineae, subtribe
Melisseae
Tribe Satureieae, subtribe
Melissinae
Tribe Saturejeae, subtribe
Melissinae
N.I. not indicated
Palynological evolutionary trends within the tribe Mentheae 97
123

Table 2 Overview of major palynological characters of all species studied within Menthinae
Species P (lm) E (lm) Shape AI CL (lm) Type OW/NW Figures
O SO OS S PS SP P
Acanthomintha ilicifolia 24.5–25.4–26.0 25.4–29.4–32.2 - ??? -??- - / 20.2–21.6–23.2 II-2 NW
Acanthomintha obovata 25.3–27.1–28.7 23.7–26.0–27.1 --?-??--0.12–0.17 21.8–23.7–25.8 II-2 NW
Blephilia ciliata 22.3–23.4–24.8 24.8–26.5–28.3 -???----0.11–0.17 18.3–19.7–21.7 II-1 NW 1j
Blephilia hirsuta 22.2–24.5–27.5 18.4–25.0–31.0 -???-??-/ 18.7–21.1–24.7 II-1 NW
Bystropogon canariensis 18.4–19.0–19.2 18.5–21.2–22.6 -??--?--/ 14.7–15.4–16.0 II-1 OW
Bystropogon canariensis var. smithianus 17.8–19.0–19.7 19.5–20.1–21.0 --??----0.17–0.22 15.4–16.0–17.0 II-1 OW
Cleonia lusitanica 31.4–39.6–43.3 30.2–36.0–40.2 - ? ?? - ? ?? ? 0.15–0.23 26.3–34.0–37.1 I-2 OW 2d
Clinopodium vulgaris 30.3–31.3–33.1 29.6–33.7–36.9 -???-?--0.18–0.23 24.2–24.6–24.9 II-2 OW
Conradina canescens 20.3–22.1–23.5 23.7–26.7–28.7 ???-----0.13–0.20 17.0–18.7–20.0 II-2 NW 1k
Conradina grandiflora 25.1–29.3–32.0 27.5–28.6–31.0 --?-??--/ 22.4–25.2–28.2 II-2 NW
Cuminia eriantha var. fernandezia 28.3–31.5–34.3 25.2–27.1–28.2 ----???-0.19–0.24 24.2–25.9–28.2 III NW
Cunila origanoides 22.0–23.4–25.0 22.0–26.3–28.6 -??-?--/ 18.4–19.8–21.4 II-1 NW
Cyclotrichium origanifolium 23.6–27.9–33.9 22.6–25.8–27.6 --?-???-/ 19.5–23.6–30.5 I-2 OW 1f
Dicerandra christmanii 29.4–30.6–31.6 29.7–33.8–36.1 -????-?--0.14–0.18 24.6–26.3–28.0 II-2 NW
Dicerandra odoratissima 22.5–24.4–27.0 26.9–28.0–29.2 -???-?--/ 18.3–20.4–23.1 II-2 NW
Glechon marifolia 25.2–31.6–34.4 25.0–32.2–37.0 ? ? ?? - ? ?? - 0.14–0.22 22.1–26.9–29.0 II-2 NW 1d
Gontscharovia popovii 24.9–29.6–32.5 27.4–32.1–36.0 -???----0.12–0.18 21.1–24.9–28.0 I-1 OW
Hedeoma ciliolata 24.8–29.8–31.1 32.2–36.0–38.7 -???----0.15–0.24 20.5–25.1–28.0 II-1 NW
Hesperozygis nitida
25.9–28.4–30.1 28.7–33.9–37.5 -????---/ 21.0–23.6–26.0 II-2 NW 1h
Heterolamium debile 30.4–36.8–43.2 27.0–31.9–35.9 --?-????0.17–0.23 23.2–28.7–32.0 I-3 OW 1e
Heterolamium debile 25.7–31.8–37.8 27.0–34.2–39.7 -????-?--0.16–0.24 21.1–24.6–30.0 I-3 OW
Hoehnea epilobioides 30.0–30.4–31.0 20.9–26.8–34.9 -??---???0.15–0.22 20.9–23.2–27.0 II-1 NW
Hoehnea minima 26.3–29.4–33.5 30.4–31.9–34.2 -???-?--/ 22.0–24.2–26.4 II-1 NW 4a
Horminum pyrenaicum 20.8–28.3–34.3 22.3–30.8–35.6 -???----0.20–0.27 17.3–23.5–28.7 I-1 OW
Hyssopus officinalis 21.1–21.7–23.1 23.5–25.9–27.3 -???----0.10–0.21 17.6–18.8–19.6 I-2 OW 1l, 2c, 3b, d
Melissa flava 29.0–31.5–35.7 31.0–36.1–39.1 -???----0.11–0.24 25.5–27.3–29.2 I-1 OW
Melissa officinalis 26.6–30.9–35.1 26.9–30.3–33.8 - - ?? - ?? ? - 0.12–0.19 23.5–26.4–30.2 I-1 OW 2b
Mentha pulegium 13.0–15.0–16.8 11.8–12.5–13.8 - - ?? - ? ?? - / 10.6–11.8–12.8 II-2 OW
Micromeria marginata 25.0–26.6–28.9 32.7–35.2–39.1 ?? ?? ? - - - - 0.10–0.17 20.1–22.4–24.3 II-1 OW 1a
Minthostachys molis 24.3–26.7–30.0 30.8–24.9–30.1 - ? ? - ?? ?? - / 18.9–21.2–22.7 II-1 NW 2f
Monarda fistulosa 30.6–32.3–34.6 31.5–37.4–41.3 -???-?--0.16–0.24 24.9–27.6–29.8 III NW 1b, 2k
Monarda punctata 35.9–39.1–42.9 31.7–41.4–47.8 -??-?--0.12–0.20 29.9–33.3–36.8 II-1 NW 2l
Monardella macrantha 26.4–27.5–28.6 31.7–33.7–36.3 -??-----0.12–0.19 22.8–24.0–25.1 I-1 NW 4d
Monardella nana 21.8–22.9–24.0 25.1–27.3–29.6 -???----0.12–0.18 18.4–20.0–21.4 I-1 NW
98 H.-K. Moon et al.
123

Table 2 continued
Species P (lm) E (lm) Shape AI CL (lm) Type OW/NW Figures
O SO OS S PS SP P
Monardella odoratissima 21.4–22.6–24.0 25.4–26.1–27.4 -???----0.14–0.21 17.3–19.1–21.8 I-1 NW 1e, h
Neoplingia leucophylloides 18.6–21.4–23.3 22.7–27.5–29.6 ???--?--/ 13.9–17.6–19.2 II-1 NW
Obtegomeria caerulescens 22.7–24.3–26.1 26.8–27.6–28.0 -????----0.16–0.21 20.0–21.4–23.4 III NW
Origanum rotundifolium 22.5–25.4–28.9 17.2–19.4–25.8 ----?????/ 16.5–18.4–19.4 II-2 OW
Pentapleura subulifera 18.1–21.4–23.8 17.9–23.0–26.8 ?? ? ? - ? ? - 0.14–0.25 15.3–18.3–21.1 II-2 OW
Piloblephis rigida 18.8–20.2–21.1 22.6–23.8–24.8 -???----/ 14.6–17.0–18.6 II-2 NW 2j, 4e
Pogogyne douglasii 25.4–27.2–30.9 26.0–28.8–30.9 --??-?--/ 22.1–23.7–25.7 II-2 NW
Pogogyne serpylloides 24.7–25.1–25.6 26.7–28.2–29.8 -???----0.20–0.29 19.7–20.8–22.0 II-2 NW
Poliomintha incana 26.5–28.6–30.0 30.1–31.1–33.2 -???----0.15–0.25 21.8–24.3–26.2 II-1 NW
Poliomintha longiflora 25.3–29.1–33.8 30.2–32.6–36.5 ????-?--0.14–0.17 20.7–24.7–28.8 II-1 NW 2g
Prunella vulgaris 26.5–31.1–35.5 19.4–22.4–25.8 ----?????/ 18.0–23.3–26.3 I-3 OW
Pycnanthemum albescens 22.8–24.2–25.2 25.1–28.0–30.5 -????----0.16–0.21 17.3–19.9–21.5 II-1 NW 1g
a
Rhabdocaulon coccineum 29.3–33.8–39.0 33.2–41.0–44.4 ????----0.15–0.21 25.4–28.6–34.5 II-2 NW
Rhabdocaulon strictum 21.8–24.7–27.0 14.7–17.6–20.3 ----????/ 15.7–18.9–21.0 II-2 NW
Rhododon ciliatus 19.9–20.7–21.7 21.8–23.7–25.2 -????----0.18–0.23 16.1–17.6–18.3 III NW
Saccocalyx satureioides 20.6–22.5–23.5 22.7–25.5–27.1 -???-?--0.18–0.26 16.6–18.6–20.1 II-1 OW 1i
Satureja thymbra 28.4–29.5–30.5 34.5–35.7–36.9 -????----/ 23.9–24.8–26.4 II-1 OW 3c
b
Stachydeoma graveolens 22.7–25.5–28.9 24.0–28.6–32.5 -????-?--0.12–0.19 18.4–22.0–26.0 II-2 NW 2i, 4c
Thymbra spicata 22.2–24.6–26.1 28.1–29.5–31.6 -???----/ 19.6–21.4–23.4 I-2 OW
Thymbra spicata 27.0–27.9–30.5 31.9–35.0–39.0 -??-----0.15–0.21 22.4–23.6–25.4 I-2 OW
Thymus pallasianus 20.7–23.2–26.7 25.2–29.0–36.8 ????----0.11–0.20 17.7–19.7–22.4 I-1 OW
Thymus serpylum 23.5–27.5–30.1 27.4–32.3–37.9 ??????---0.13–0.18 21.0–23.8–27.0 I-1 OW 2a, 3e
Zataria multiflora 17.2–19.0–20.9 18.6–19.5–21.6 - ? ?? - ?? - - / 12.8–15.1–16.4 II-1 OW 3a
Ziziphora clinopodioides 24.2–25.8–27.2 29.9–32.3–35.3 ???-----0.14–0.24 20.4–22.2–24.8 I-1 OW 1c, 4b, f
Numbers refer to minimum–mean–maximum except for AI (apocolpium index) values that correspond to a range
P polar axis, E equatorial diameter, O oblate, SO suboblate, OS oblate spheroidal, S spherical, PS prolate spheroidal, SP subprolate, P prolate, CL colpus length, OW Old World, NW New
World. -, absent; ?, present; ??, dominance; /, no data
a, b
Micrographs of Pycnanthemum incanum and Satureja spinosa used for references which are not included taxa in the table as lacking of enough pollen grains to calculate mean value
Palynological evolutionary trends within the tribe Mentheae 99
123

are regular and at the mesocolpia over ten secondary
lumina per primary lumen were counted. The secondary
lumen size and number decreases towards the poles and
apertures. Observed in: Cleonia, Cyclotrichium, Hyssopus,
Thymbra (Fig. 2c, d). Type I-3: The primary muri are
irregular in pattern and sometimes discontinuous. Each
primary lumen contains more than 20 rounded secondary
lumina. Observed in: Heterolamium, Prunella (Fig. 2e).
Type II: microreticulate
Two subtypes are recognized based on the possible pres-
ence of tectal connections in the lumina. Type II-1:
Microreticulate without secondary tectal connections.
Observed in: Blephilia, Bystropogon, Cunila, Hedeoma,
Hesperozygis, Hoehnea, Micromeria, Minthostachys,
Monarda punctata, Neoplingia, Poliomintha, Pycnanthe-
mum, Saccocalyx, Satureja, Zataria (Fig. 2f–h). Type II-2:
The basic exine ornamentation is microreticulate, but sec-
ondary tectal connections are present in this subtype. Some
perforations are elongated and subdivided into two or three
smaller units by muri at a slightly lower plane. Observed
in: Acanthomintha, Clinopodium, Conradina, Dicerandra,
Glechon, Mentha, Origanum, Pentapleura, Piloblephis,
Pogogyne, Rhabdocaulon, Stachydeoma (Fig. 2i, j).
Type III: perforate
The exine ornamentation is perforate. The distance
between the edges of adjacent perforations is greater than
the diameter of perforations. Observed in: Cuminia,
Rhododon, Monarda fistulosa and Obtegomeria (Fig.
2k).
Pollen wall stratification and ultrastructure
All taxa studied show the same exine stratification (Fig. 3).
The tectum is thicker than the foot layer. Columellae are
simple, unbranched and densely spaced. The foot layer is
continuous or distinctly discontinuous (Fig. 3c–e), rela-
tively thin and supported by a very thin, often hardly
discernible endexine (Fig. 3c–e). The intine is thicker
below the colpi than at the mesocolpia (Fig. 3b, e). Pol-
lenkitt is accumulated in the infratectum (Fig. 3e).
Fig. 1 SEM micrographs of
pollen grains of Menthinae. a–f
Variation of pollen shape in the
equatorial view. g–l Polar view
with variations of shape and
aperture number. a Oblate
pollen grain with elliptic outline
of Micromeria marginata.
Suboblate pollen grain,
b Monarda fistulosa,
c Ziziphora clinopodioides.
d Oblatespheroidal pollen grain
with circular outline, Glechon
marifolia. Monardella
odoratissima, e oblate
spheroidal pollen, h circular
outline. f Subprolate pollen
grain of Cyclotrichium
origanifolium. g Pentacolpate
pollen grain of Pycnanthemum
incanum. Hexacolpate pollen
grain with circular shape,
h Monardella odoratissima,
i Saccocalyx satureioides,
j Blephilia ciliata. Hexacolpate
pollen grain with elliptic shape,
k Conradina canescens,
l Hyssopus officinalis
100 H.-K. Moon et al.
123

Orbicules
Orbicules were absent in all species investigated (Fig. 4).
The inner locule wall displays a characteristic annular or
star shaped pattern because of the prominent underlying
endothecium thickenings (Fig. 4d). The tapetal membrane
is completely smooth without any sporopollenin deposition
(Fig. 4d–f).
Discussion
Menthinae are a stenopalynous group. Their pollen is
small to medium size (P = 13.0–43.3 lm), hexacolpate
(mixed with few pentacolpate pollen grains in Prunella
vulgaris and Pycnanthemum incanum) with an oblate
to prolate shape, and a perforate, microreticulate or
bireticulate exine ornamentation. The colpus membranes
are beset with granules and the exine stratification typi-
cally shows simple columellae. Orbicules are consistently
absent.
Pollen features of Menthinae compared to other
members of tribe Mentheae
There is ample evidence in the literature that the size of
pollen grains is greatly affected by different preparation
treatments and generally critical point-dried pollen is
smaller than acetolysed pollen grains in SEM (Harley 1992;
Lens et al. 2005; Moon et al. 2008a, b; Reitsma 1969;
Schols et al. 2004). The present results are based on critical
point dried anthers in order to conserve optimally the nat-
ural size and shape. Although the size varies between taxa
of Menthinae, it is notable that the range of size variation is
more or less constant within the same genus (Table 2).
In Menthinae, we found some pentacolpate pollen grains
in Prunella vulgaris and Pycnanthemum incanum.This
aperture heteromorphism was known in 17 species
belonging to eight genera in tribe Mentheae (hexacolpate is
always dominant but mixed together with tetra-, penta- or
octocolpate pollen; Moon et al. 2008a, b; Trudel and
Morton 1992). Even if aperture heteromorphism might
offer an evolutionary advantage due to an increased
Fig. 2 SEM micrographs of
variation of exine
ornamentation in Menthinae.
Variations of bireticulate exine
ornamentation; a Thymus
serpyllum, b Melissa officinalis,
c Hyssopus offiicinalis, d
Cleonia lusitanica, e
Heterolamium debile. Reticulate
exine ornamentation; f
Minthostachys mollis, g
Poliomintha longiflora, h
Hesperozygis nitida. Reticulate
exine ornamentation with
possible secondary tectal
connections visible; i
Stachydeoma graveolens, j
Piloblephis rigida. Variation of
exine ornamentation in the
genus Monarda; k perforate—
Monarda fistulosa, l reticulate—
Monarda punctata
Palynological evolutionary trends within the tribe Mentheae 101
123

germination success (Dajoz et al. 1995; Furness and Rudall
2004; Mignot et al. 1994), it occurs rarely and only a very
limited number of pollen grains are affected (less than 5%).
In addition, aperture heteromorphism occurs often in cul-
tivated individuals (A.J. Paton, personal communication),
and it could therefore be induced by atypical ecological
circumstances. Our results show that all taxa studied
in Menthinae have predominantly hexacolpate pollen,
which supports hexacolpate pollen as a synapomorphy for
Nepetoideae. Simple columellae are plesiomorphic and
found in most gynobasic-styled Labiatae (Abu-Asab and
Cantino 1992). All taxa studied in Menthinae have simple
columellae and a continuous or discontinuous foot layer
with hardly observable endexine (Fig. 3c–e). Such an exine
stratification also occurs in subtribes Salviinae and Ne-
petinae, and is common in subfamily Nepetoideae (Harley
1992; Harley et al. 1992; Moon and Hong 2003; Moon
et al. 2008a, b; Nabli 1976).
Fig. 3 SEM and TEM micrographs of pollen wall of Menthinae.
a Cross section through exine of Zataria multiflora. b–h TEM
observations of the ultrastruture of the pollen wall. b Exine thickness
decreases towards the apertures (A) while the intine is thicker below
the colpi than at the mesocolpia; Hyssopus officinalis. c–e Magnified
part of the pollen wall showing tectum (T), columellae (C), foot layer
(F), endexine (E) and intine (In). c Foot layer is discontinuous and
endexine is hardly divided with foot layer; Satureja spinosa. d Foot
layer is continuous; Hyssopus officinalis. e Exine is thicker below the
mesocolpia than at the apertures (A) and deposition of pollenkitt
(black arrows) in the infratectum; Thymus serpyllum
Fig. 4 SEM micrographs of
Menthinae. a Anther with two
thecae of Hoehnea minima.
b The completely dehisced
stamen showing the inner locule
wall without orbicules;
Ziziphora capitata. c Structure
of the endothecium thickenings
in cross section of Stachydeoma
graveolens. d Inner locule wall
with no traces of orbicules of
Monardella macrantha, left;
inner locule wall, right; detail of
star-shaped endothecium cells.
Detail of annular shaped
endothecium cells without
orbicules; e Piloblephis rigida,
f Ziziphora clinopodioides
102 H.-K. Moon et al.
123

Exine ornamentation variation in Menthinae
The observed variation in exine ornamentation could be
defined as bireticulate, microreticulate and perforate.
Microreticulate exines are most common in Menthinae
while a bireticulum is common in Salviinae and Nepetinae
and in other Lamiaceae as well (Abu-Asab and Cantino
1992, 1994; Jamzad et al. 2000; Moon et al. 2008a, b;
Trudel and Morton 1992; Wagstaff 1992). The shared
subtype of bireticulate exine suggests a close relationship
between Cleonia and Cyclotrichium, Hyssopus and Thym-
bra, while Gontscharovia has a similar exine pattern as
Horminum, Monardella, Thymus and Ziziphora. Histori-
cally, Cleonia was considered related with Prunella
(Table 1; Briquet 1897; Wunderlich 1967). In addition the
molecular phylogeny suggested Horminum as a sister
genus of Cleonia and Prunella (Walker and Sytsma 2007).
The exine type corroborates this assumption, since all three
genera possess bireticulate pollen even though we can find
slight intergeneric differences in ornamentation (type I;
Fig. 2d, e). Minthostachys has once been included in Bys-
tropogon (Table 1; Bentham 1876; Briquet 1897); their
similar exine ornamentation indeed confirms a close rela-
tionship between these genera. A similar example could be
found in the genera Poliomintha, Rhabdocaulon and
Stachydeoma, which possess a similar exine as Hedeoma
(Table 1). Perforate exine ornamentations were found in
Cuminia, Obtegomeria, Rhododon and Monarda fistulosa.
It should be noted that bireticulate pollen with 10–20
secondary lumina is restricted to Old World taxa (Cleonia,
Cyclotrichium, Hyssopus, Thymbra) while perforate pollen
was found only in New World taxa (Cuminia, Monarda
fistulosa, Obtegomeria, Rhododon; Table 2). In fact, bire-
ticulate pollen occurs generally in Old World taxa except
for the New World genus Monardella (Table 2).
At generic level, exine sculpturing appears as a stable
character, except for Monarda (Table 2; Fig. 2k, l). The
variation in exine patterns found in Monarda seems to
correlate with its subgeneric classification. Monarda
fistulosa (subgenus Monarda) for instance possesses per-
forate grains and Monarda punctata (subgenus Cheilyctis)
reticulate grains (Fig. 2k, l; Prather et al. 2002). However,
our sampling for the genus Monarda is insufficient (2/20
species) to address the infrageneric relationships based on
pollen data alone.
Systematic implications of exine ornamentation
at tribal level
The most recent accepted classification of tribe Mentheae
(Harley et al. 2004) included many genera from different,
previously recognized tribes (Table 1). Although tribe
Mentheae is a well-supported monophyletic group, the
subtribal delimitation and the relationships between genera
are still poorly resolved (Cantino 1992; Harley et al. 2004;
Trusty et al. 2004; Wagstaff et al. 1995; Walker and
Sytsma 2007). In the most recent phylogenetic hypothesis
produced by Walker and Sytsma (2007), subtribe Salviinae
appears as a monophyletic group. However, subtribe
Menthinae splits into two major lineages and genus Lyco-
pus of the Menthinae is sister to subtribe Nepetinae. It
should be mentioned that this molecular study was focused
on genus Salvia with related genera and includes only 35
out of 65 Mentheae genera (Walker and Sytsma 2007).
Nevertheless, to date this phylogeny is the most compre-
hensive one of the Mentheae (Walker and Sytsma 2007;
Fig. 5).
The different exine patterns recognized evolved inde-
pendently several times throughout the Mentheae (Fig. 5).
Subtribe Salviinae possesses mainly bireticulate pollen
except for Lepechinia and Chaunostoma, which have per-
forate pollen. The perforate exines of Lepechinia and
Chaunostoma of Salviinae support that these genera are
sister to all other genera within Salviinae (Epling 1948;
Moon et al. 2008a). According to molecular topologies,
Salvia is not a monophyletic group (Walker et al. 2004;
Walker and Sytsma 2007) and pollen characters provide
further evidence for this finding (Moon et al. 2008a).
Interestingly bireticulate exines with thin primary muri or
with a secondary reticulum with large lumina are restricted
to Salvia clade I (Moon et al. 2008a). In addition, these
features could be interpreted as more derived based on a
reduced tectal surface (Walker and Doyle 1975). Reticulate
exine patterns occur mainly in Menthinae clade I. The
sister group of Menthinae clade I consists of subtribe Ne-
petinae and Menthinae clade II (Lycopus, Cleonia,
Prunella and Horminum). Menthinae clade II has prob-
lematic subtribal delimitations. Trusty et al. (2004)
demonstrated conflicting results with traditional taxonomy
showing that Hyssopus (Menthinae) is closely related with
Lallemantia (Nepetinae), while Horminum (Menthinae),
Nepeta (Nepetinae) and Prunella (Menthinae) grouped
together based on macromolecular data. The shared bire-
ticulate exine ornamentation supports that Menthinae clade
II and Nepetinae clade are closely related (Fig. 5). The
taxonomic position of Lycopus has been problematic
because of contradictory placements in different molecular
phylogenies (Bra
¨
uchler et al. 2005; Trusty et al. 2004;
Wagstaff et al. 1995; Walker and Sytsma 2007). For
instance, the results of cpDNA restriction site analyses by
Wagstaff et al. (1995) showed that Lycopus is sister to the
major Menthinae clade, while combined analysis of the
chloroplast regions trnL-F, psbA-trnH and the nuclear
rDNA ITS suggested
Lycopus as a sister of the Nepetinae
clade (Walker and Sytsma 2007). However, the palyno-
logical data of Lycopus provide evidence for a relationship
Palynological evolutionary trends within the tribe Mentheae 103
123

with Menthinae clade I (Moon and Hong 2003). Unfortu-
nately, the present molecular framework of Lamiaceae has
a very poor sampling of Mentheae taxa and particularly the
monophyly of Nepetinae awaits confirmation. Even within
Nepetinae the relationships among genera are conflicting
(Bra
¨
uchler et al. 2005; Jamzad et al. 2003; Trusty et al.
2004; Wagstaff et al. 1995; Walker et al. 2004; Walker and
Sytsma 2007). From a palynological point of view it is
interesting to note that bireticulate exine patterns in
Salviinae show a clear distinction between primary and
secondary reticula, while bireticulate pollen of Nepetinae
has a rather vague distinction between the two incongruent
reticula (Moon et al. 2008a, b).
Genera of uncertain affinity
The present study included two genera of uncertain sub-
tribal affinity, Heterolamium and Melissa. Heterolamium is
Fig. 5 Diagrams of different exine patterns in Mentheae and their
distribution on the most recent molecular phylogenetic tree (simpli-
fied tree based on Walker and Sytsma 2007; their Figs. 3, 4). The
three major types of exine ornamentation recognized were plotted on
the topology; their occurrence in each clade is indicated as follows
without reference to individual species (white bireticulate, black
reticulate, stripe perforate). The taxa indicated by an asterisk were
included in the present study but lacking in Walker and Sytsma
(2007)—their phylogenetic position is estimated by assuming that
genera are monophyletic
104 H.-K. Moon et al.
123

endemic to China and historically this genus belonged to
Orthosiphon. However, Orthosiphon debile Hemsl. has
been accepted as an independent genus Heterolamium
based on its distinct anther and corolla characters (Li and
Hedge 1994). According to the most recent classification of
Lamiaceae (Harley et al. 2004) Heterolamium belongs to
tribe Mentheae without any indication of its subtribal
position. Heterolamium has bireticulate pollen with dis-
continuous primary lumina consisting of over 20 secondary
lumina (type I3, Fig. 2e). Although Prunella possesses the
same exine type as Heterolamium, Prunella belongs to the
problematic Menthinae clade within subtribe Nepetinae.
Furthermore, its exine ornamentation resembles species of
Drepanocaryum, Lophanthus and Nepeta, which are all
members of Nepetinae (Moon et al. 2008b). In addition,
Heterolamium is characterized by 15 calyx nerves and four
stamens with the posterior pair longer than the anterior,
both features being synapomorphies of Nepetinae (Harley
et al. 2004). In conclusion, our results strongly support a
position for Heterolamium in subtribe Nepetinae.
The genus Melissa consists of four species, distributed
through Europe, North Africa, Macaronesia and Asia.
Melissa was previously placed in subtribe Melissinae of
tribe Satureieae together with several other genera, which
currently belong to subtribe Menthinae (Table 1; Bentham
1876; Briquet 1897; Wunderlich 1967). Recent molecular
phylogenies suggest Melissa as a member of subtribe
Salviinae in Mentheae (Trusty et al. 2004; Walker and
Sytsma 2007). Melissa has bireticulate pollen grains. The
primary muri are thick with rounded primary lumina and
the number of secondary lumina is 5–10 per primary
lumen (Fig. 2b). This kind of exine ornamentation is very
similar to the ornamentation pattern in the genera Dorys-
taechas, Meriandra, Perovskia and Rosmarinus of the
subtribe Salviinae (Moon et al. 2008a), supporting the
hypothesized relationship between these genera (Walker
et al. 2004; Walker and Sytsma 2007). Thus, palynological
data support the inclusion of Melissa into the Salviinae.
However, within subtribe Menthinae, a similar exine
ornamentation was found in taxa from genera Gontscha-
rovia, Horminum, Monardella, Thymus and Ziziphora
(Table 2).
Lack of orbicules
In general, the presence of orbicules is considered as a
plesiomorphic trait. Indeed, in angiosperms orbicules are
mainly restricted to species possessing a secretory tape-
tum, which is predominantly present in the early
diversified lineages of flowering plants (i.e., Furness and
Rudall 1998; Huysmans et al. 1998). In Chloanthaceae
(currently tribe Chloantheae of Lamiaceae sensu Harley
et al. 2004), variation in shape and surface patterns of
orbicules was found to be taxonomically useful (Raj and
El-Ghazaly 1987). All genera investigated of Mentheae
are lacking orbicules (Moon et al. 2008a, b). The locule
wall is smooth, with no traces whatsoever of orbicule-
like sporopollenin deposition. The absence of orbicules
in Mentheae and their presence in tribe Chloantheae
actually confirms the plesiomorphic nature since tribe
Chloantheae belongs to the basal Lamiaceae subfamily
Prostantheroideae (Harley et al. 2004; Raj and El-Ghaz-
aly 1987). However, a study on Lavandula dentata (tribe
Ocimeae of subfamily Nepetoideae sensu Harley et al.
2004; Suarez-Cervera and Seoane-Camba 1986) showed
that this species has a secretory tapetum without orbicule
formation. The tapetum type of Mentheae is unknown
and therefore further ontogenetical studies of both pollen
and tapetal cells will be necessary to provide insight in
the relationship between tapetum type and orbicule
occurrence.
Conclusion
Mentheae is a stenopalynous group with hexacolpate
pollen, perforate/microreticulate/bireticulate exine orna-
mentation and a pollen wall stratification with simple
unbranched columellae. Variation in exine ornamentation
may have systematic importance particularly at generic
level. Our results suggest Heterolamium to be a member of
subtribe Nepetinae and Melissa to belong to subtribe Sal-
viinae. The exine variation observed in Monarda might
have potential taxonomic value at subgeneric level. How-
ever, we found variability in some pollen characters
between closely related species and a remarkable associa-
tion between specific exine ornamentation types and the
major geographic areas. A phylogenetic study of Mentheae
(combining morphology and macromolecules) based on a
relevant sampling will be necessary to illuminate the
enigmatic intergeneric relationships. The present study
provides pollen morphological characters that hopefully
will be added in future phylogenetic analyses.
Acknowledgments We thank the directors of the herbaria of BR, G,
GH, K, LV, MO and S for permitting the examination of specimens,
either through loans or during visits. Sincere thanks also to two
anonymous reviewers for their critical reading of manuscript and
helpful suggestions. We are also grateful to Anja Vandeperre
(K.U.Leuven) and An Vandoren (TEM, K.U.Leuven) for technical
assistance. H.K. Moon thanks Dr. Alan Paton (Royal Botanic Gar-
dens, Kew) for valuable discussions and Dr. Frederic Lens
(K.U.Leuven) for helpful comments that improved this manuscript. A
personal research grant of the K.U.Leuven (DB/06/054) was awarded
to H.K. Moon. The Fund for Scientific Research-Flanders (FWO,
G.0268.04 and G.0250.05) and the K.U.Leuven (OT/05/35) finan-
cially supported this research. S. Vinckier was a postdoctoral fellow
of the Fund for Scientific Research-Flanders (FWO) during the course
of this study.
Palynological evolutionary trends within the tribe Mentheae 105
123

Appendix
Table 3
Table 3 Voucher specimens of subtribe Menthinae that are examined in the present study
Species Voucher specimens
Acanthomintha ilicifolia A.Gray USA, 26–29.05.1915. Dutton & Walker 3819 BR
Acanthomintha obovata Jeps. USA, 18.05.1919. Walker 5094 BR
Blephilia ciliata (L.) Benth. USA, 13.06.1966. Radford 44758 BR
Blephilia hirsuta (Pursh) Benth. USA, 12.07.1966. Radford 44922 BR
Bystropogon canariensis (L.) L’He
´
r Canary Is., without date, Bullemont 1855 BR
Bystropogon canarensis var. smithianus H.Christ Canary Is., 15.05.1901. Bornmu
¨
ller 2765 BR
Cleonia lusitanica (L.) L. Morocco, 13.05.1934. Wall 45 S
Clinopodium vulgare L. Cultivated in KEW accession no.: 1994–2824
Conradina canescens A. Gray USA, 30.11.1969. Godfrey 69283 BR
Conradina grandiflora Small USA, 24.10.1956. Ahles & Bell 21395 BR
Cuminia eriantha var. fernandezia (Colla) Harley Chile, 23.11.1991. Billiet & Jadin 5631 BR
Cunila origanoides (L.) Britton USA, 07.09.1897. Anonymous 323b BR
Cyclotrichium origanifolium (Labill.) Manden. Lebanon, 06.07.1897. Bornmu
¨
ller 1260, BR
Dicerandra christmanii Huck & Judd USA, 11.09.1987. Skean, Jr. 2130 MO
Dicerandra odoratissima R.M.Harper USA, 17.09.1967. Radford & Leonard 11479 BR
Glechon marifolia Benth. Uruguay, 12.02.1955. Pedersen 3627 BR
Gontscharovia popovii (B. Fedtsch. & Gontsch.) Boriss. URSS, 31.08.1931. Anonymous 206 K
Hedeoma ciliolata (Epling & W.S.Stewart) R.S.Irving Mexico, 01.10.1954. Rzedowski 5003 GH
Hesperozygis nitida (Benth.) Epling. Brazil, 22.09.1976. Dombroswski 6442 K
Heterolamium debile (Hemsl.) C. Y. Wu China, 03.1889. Henry s.n. K
China, 1885–1888. Henry 5770 GH
Hoehnea epilobioides (Epling) Epling Brazil, 23.10.1974. Kummrow 688 K
Hoehnea minima (Schmidt) Epl. Brazil, 26.01.1916. Duse
´
n 17542 GH
Horminum pyrenaicum L. Italy, 26.06.1969. Cnops 21169 BR
Hyssopus officinalis L. Spain, 22.09.1974. Bondı
´
a et al., 1242 GF BR
Cultivated in KEW accession no.: 1975–1170 K
a
Melissa flava Benth. India, without date, Strachey and Winterbottom 1 BR
Melissa officinalis L. France, 1986. Sotiaux s.n. BR
Spain, 1869. Bourgeau s.n. BR
a
Cultivated in KEW accession no.: 1994–2690 K
a
Mentha pulegium L. Cultivated in KEW accession no.: 1994–1897 K
a
Micromeria marginata (Sm.) Chater Cultivated in KEW accession no.: 1995–1960 K
a
Minthostachys mollis Griseb Ecuador, 10.08.1939. Asplund s.n. BR
Monarda fistulosa L. USA, 06.08.1973. Bouharmont 8498. BR
Monarda punctata L. USA, 26.10.1957. Ahles & Haesloop 38096 BR
Monardella macrantha A.Gray Cultivated in KEW accession no.: 1980–998 K
a
Monardella nana A. Gray Cultivated in KEW accession no.: 1999–270 K
a
Monardella odoratissima Benth. USA, 29.08.1969. Howell 46064 BR
Neoplingia leucophylloides Ramamoorthy Mexico, 05.08.1982. Hiriart & Medrano 12792 K
Obtegomeria caerulescens (Benth.) Doroszenko USA, 16.08.1986. Hermes, Cuadrov & Gentry 2706 MO
Origanum rotundifolium Boiss. Cultivated in KEW accession no.: 1968–19106 K
a
Pentapleura subulifera Hand.-Mazz. Iraq, 4–9. 07. 1957. Rechinger 12085 K
Piloblephis rigida (Bartram ex Benth.) Raf. USA, 13.02.1995. Holst et al. 4543 MO
Pogogyne douglasii Benth. USA, California, 31.05.1892. Bioletti s.n. BR
106 H.-K. Moon et al.
123

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Pogogyne serpylloides (Torr.) Gray USA, California, 28.04.1964. Rose 64044 BR
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a
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Satureja spinosa L. Cultivated in KEW accession no.: 1989–3009 K
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Stachydeoma graveolens (Chapm. ex A. Gray) Small USA, 18.08.1962. Godfrey 62494 BR
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Thymbra spicata L. Italy, 18.06.1883. Ascherson 470 BR
Cultivated in KEW accession no.: 2001–825 K
a
Thymus pallasianus Cultivated in KEW accession no.: 2001–4194 K
a
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a
Zataria multiflora Boiss. Iran, 16.05.1892. Bornmu
¨
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Ziziphora clinopodioides Lam. Turkey, 01.09.1993. Vas
ˇ
a
´
k s.n. BR
All taxa were investigated by scanning electron microscopy
a
The selected taxa also observed by transmission electron microscopy
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