Phylogenetics and floral structure in

Thunbergioideae and Avicennia (Acanthaceae)

AGNETA JULIA BORG

Licentiate Thesis in Systematic Botany

Department of Botany
Stockholm University
2008
 2

Phylogenetics and floral structure in

Thunbergioideae and Avicennia (Acanthaceae)

AGNETA JULIA BORG

Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden

Contents:

p. 3 Introduction
p. 4 Aims
p. 5 Materials and methods
p. 5 Results and discussion
p. 6 Acknowledgements
p. 6 Literature cited

p. 8 Paper I:
Borg, A.J., McDade, L.A. & Schönenberger, J. Molecular phylogenetics and
morphological evolution of Thunbergioideae (Acanthaceae). Taxon. In press.

p. 24 Paper II:
Borg, A.J. & Schönenberger, J. Floral structure and development in Avicennia.
Manuscript.

Licentiate Thesis in Systematic Botany

Department of Botany
Stockholm University
2008

INTRODUCTION
The flowering plant family Acanthaceae
(Lamiales) consist of at least 4000 mainly
tropical and subtropical species. The vast
majority of these belong to the core
Acanthaceae (Acanthoideae sensu Scotland
& Vollesen 2001) recognised by the shared
presence of a unique kind of fruit; an
explosively dehiscent capsule with the few
seeds borne on hook-like outgrowths of the
funiculus called retinacula. In addition to the
core Acanthaceae, two small lineages, the
Thunbergioideae and Nelsonioideae sensu
Scotland & Vollesen (2001), have tradition-
ally been associated with Acanthaceae,
though their markedly different morphology
has led to various suggestions of how to
classify the constituent genera and some
authors have placed Thunbergioideae and
Nelsonioideae in distinct families. However,
the close relationship to the core Acanth-
aceae has now gained support from both
molecular and morphological studies (e.g.
Hedrén & al., 1995; McDade & Moody,
1999; Schönenberger & Endress 1998).
More recently and quite surprisingly, a
molecular study by Schwarzbach & McDade
(2002) using data from both the chloroplast
and the nuclear genome, implied that the
black mangrove genus Avicennia, usually
treated as a separate family in Lamiales or as
a genus within Verbenaceae, is also part of
Acanthaceae. In their study, Avicennia is
consistently placed as sister group to
Thunbergioideae, albeit with weak support.
Although there seems presently to be
general agreement regarding the delimitation
of the family as a whole, as well as of the
major lineages, the exact relationships
among lineages and among and within the
genera of the three smaller lineages have
been little studied and are still not well
understood. This licentiate thesis focuses on
Thunbergioideae and Avicennia.
3
Thunbergioideae
The Thunbergioideae comprise five
genera, the largest of which, Thunbergia,
contains about 100 species restricted to
tropical and subtropical regions of Africa,
Madagascar, Asia, and Australia. The second
largest genus, Mendoncia (ca. 60 species), is
most diverse in Central and South America
with some African representatives and a few
species native to Madagascar. The remaining
genera are Pseudocalyx with ca. 7 species in
Africa and Madagascar, and monotypic
Anomacanthus in tropical Africa, and
monotypic Meyenia in India. The subfamily
is characterized by a predominantly twining
habit, enlarged bracteoles, and a reduced
calyx. Furthermore Thunbergioideae lack the
retinaculate fruits found in core Acanth-
aceae.
There are no existing subgeneric classi-
fications for Mendoncia or Pseudocalyx (but
see Profice, 1988, for Mendoncia in Brazil).
More attention has been directed towards
Thunbergia. Lindau (1893) subdivided
Thunbergia into four sections based on
morphology and arrangement of flowers.
Bremekamp (1955) revised and extended
Lindau’s subdivision and proposed eight
subgenera. His classification largely concurs
with a recent study of floral development
and structure in Thunbergia by Schönen-
berger (1999), but has never been compared
to a hypothesis of phylogenetic relationships
based on DNA sequence data. So far, no
molecular study has included more than a
couple of representatives from Thunbergia
and Mendoncia. Accordingly, the under-
standing of phylogenetic relationships within
Thunbergioideae is currently very limited.
Avicennia
The black mangrove genus Avicennia
consist of around 8 species of trees which
grow in the intertidal zone of coastal
mangrove forests, ranging widely throughout
tropical and warm temperate regions of the
world (Tomlinson; 1995). Avicennia has

always presented a problem to systematists.
Like other mangroves, Avicennia exhibits a
number of conspicuous adaptations to the
mangrove habitat of which they are an
important constituent. These include
specialized erect, lateral roots developed as
pneumatophores, longitudinal air chambers
in the cortex of the pneumatophores, toler-
ance to high salt concentrations, xerophytic
leaves with well-developed hypodermis and
viviparous fruits which are sea-water
dispersed. To a large extent, morphological
characters in Avicennia appear to be
controlled by environmental factors (Duke &
al., 1998), making it a difficult task to trace
the evolutionary history of the genus.
However, there are also a number of traits,
involving stem and root anatomy, pollen
morphology, gynoecial anatomy, and
embryology, which may or may not be
related to the mangrove habit (Sanders,
1997). Studies of these characters have led to
a number of different suggestions of how to
classify Avicennia: E.g., Bentham & Hooker
(1876) placed Avicennia in Verbenaceae,
Van Tieghem (1898) suggested a position
near Santalales, Croizat proposed a place
among Dipterocarpaceae (letters from
Croizat to Moldenke, in Moldenke, 1960)
and Dahlgren (1975) placed the genus in
Celastraceae.
Although several authors have given
attention to the morphology of particular
organs of Avicennia (e.g. pollen: Erdtman,
1945 and Saxena, 1981; embryology:
Padmanabhan, 1964 and Maheshwari &
Kapil, 1966; gynoecium: Junell, 1934; wood
anatomy: Zamski, 1979 and Carlquist,
1992), no comprehensive study of floral
morphology and development of the genus
has been presented to date, and morpho-
logical support for a relationship to other
Acanthaceae are still lacking. The need to
study Avicennia in more detail in order to
learn more about the taxonomic status and
relationships of the genus has been pointed
4
out several times (Padmanabhan, 1960;
Schwarzbach & McDade, 2002).
AIMS
The main purpose of paper I is to place
Thunbergioideae among the other acanth-
aceous lineages and to test earlier
morphology-based hypotheses about the
relationships among and within the larger
genera of the subfamily.
Paper II aims at providing a comparative
investigation of floral structure and develop-
ment in Avicennia which may help to shed
light on the evolutionary history of the genus
in relation to other Acanthaceae.
MATERIAL AND METHODS
In paper I, 30 species from the three
major genera of Thunbergioideae (Thun-
bergia, Mendoncia, and Pseudocalyx) were
included, covering the main distribution
areas of the genera. The sampled Thunbergia
species (21 species) represent all 8 sub-
genera circumscribed by Bremekamp
(1955). The sampling also includes T.
arnhemica, the only Thunbergia species
native to Australia.
Two species from each of the two tribes
of the Acanthoideae; the Acantheae and
Ruellieae (sensu Scotland & Vollesen 2000),
were sampled along with single
representatives of three out of six genera of
Nelsonioideae. Three out of 8 species of
Avicennia (sensu Tomlinson, 1995) were
included and chosen with respect to geo-
graphic distribution. The genus Schlegelia
which is possibly sister to Acanthaceae (71%
jackknife support in Bremer & al., 2002)
was chosen as outgroup. Nucleotide
sequences from three chloroplast DNA
regions (rps16, rpl16, trnT-trnL) were
produced and analysed using both parsimony
and Bayesian methods, and bootstrap

support was estimated (see paper I for
details).
In paper II, three out of eight species of
Avicennia (sensu Tomlinson, 1995) were
studied, representing the biogeographic
distribution of the genus: A. germinans
occurring along the coasts of the Americas
and the Atlantic coast of Africa, A. bicolor
occurring along the pacific coast of Central
America, and A. alba occurring along the
coasts of south-eastern Asia and islands of
the south-western Pacific. Microtome
sections of old flower buds were prepared
and studied with light microscope, and buds
were also prepared for and studied with
scanning electron microscopy (see paper II
for details).
RESULTS AND DISCUSSION
Paper I
Both parsimony and Bayesian analyses
of molecular data strongly suggest that
Thunbergioideae is monophyletic, as has
been implied in earlier molecular studies
based on more limited taxon sampling (e.g.
Hedrén & al. 1995; Scotland & al. 1995;
McDade & al. 2000). The results also agree
with the intrafamilial position of
Thunbergioideae proposed in earlier higher-
level studies (e.g. Scotland & al. 1995;
McDade & Moody, 1999; both studies did
not include Avicennia). In our analyses,
Avicennia is consistently placed as sister to
Thunbergioideae although with only
moderate support, as was also found by
Schwarzbach & McDade (2002). Acanth-
oideae is sister to Thunbergioideae plus
Avicennia, and Nelsonioideae is sister to all
other Acanthaceae. Our results further
suggest that Thunbergia and Mendoncia are
both monophyletic, and Mendoncia is sister
to Thunbergia plus Pseudocalyx. Relation-
ships within the two largest genera
Mendoncia and Thunbergia are highly
resolved and most branches are strongly
5
supported by molecular data. Also morpho-
logical characters support the results. Beside
the general characteristics of the subfamily
(see introduction), the sister taxa Thunbergia
and Pseudocalyx both have two fertile
locules in the gynoecium and the fruit is a
dry capsule, whereas in Mendoncia only one
of two initiated locules develops fully
(Schönenberger & Endress 1998) and the
fruit is a fleshy drupe. However, there are
also characters shared by Mendoncia and
Pseudocalyx, including type of anther
dehiscence, stigma shape and indumentum
(Brummitt 1989; Schönenberger & Endress
1998).
Subgeneric relationships in Mendoncia
reflect the geographic distribution of species.
The genus displays a trans-Atlantic dis-
junction, and the results from the molecular
analyses support a clade consisting entirely
of American species, and two lineages of
African species. Some of the African species
have earlier been ascribed to two isolated
genera, i.e. Monachochlamys Baker and
Afromendoncia Gilg. However, in our
analyses, neither of these two taxa turned out
monophyletic and our data therefore support
Benoist’s (1944) suggestion that the two
genera should be included in Mendoncia.
Molecular data partially support the
existing morphology-based classification of
Thunbergia proposed by Bremekamp
(1955), but not all eight subgenera are
monophyletic and the classification needs
revision. Based on reconstructions of
character-evolution in Thunbergia, we found
a twining habit to be ancestral for that genus.
The thecal awns, characteristic for many
Thunbergia species (Schönenberger, 1999),
have probably evolved from unicellular
bristles. Generally, habit, calyx morphology,
and anther dehiscence patterns seem to
reflect molecular evolutionary relationships
particularly well among Thunbergia species,
whereas the presence or absence of anther
appendages and stigma structure are
somewhat more labile and most likely are

closely correlated with differences in
pollination biology.
Paper II
Phylogenetic relationships of the small
black mangrove genus Avicennia have been
difficult to trace due to the presence of many
convergent characters (Duke & al, 1998).
Our study of early floral development and
floral structure documents a number of
similarities between Avicennia and
Thunbergioideae, including a basically
pentamerous floral ground plan with a
dimerous gynoecium, a persisting calyx, left
contort corolla aestivation, presence of five
stamen primordia of which one degenerates,
swollen filament bases, glandular hairs on
filament bases, presence of pollen sac
placentoids, collateral ovule arrangement,
and unequal stigma lobes. Other similarities
include articulated stems and fruit
morphology. However, none of these
features seems to provide a clear
synapomorphy for the clade with Avicennia
and Thunbergioideae. Several of these and
additional characters are shared with
Acanthaceae in general (e.g. corolla
aestivation, inflorescence structure and
endosperm development (Padmanabhan,
1964; Mohan Ram & Wadhi, 1965) and
some are even commonly found in various
families of the Lamiales, such as a
pentamerous floral ground plan with a
dimerous gynoecium, pollen sac placentoids,
and a strong reduction or even a complete
loss of the dorsal (median) stamen.
Earlier authors have linked Avicennia to
various groups both within and outside the
Lamiales (see introduction). Although our
results support a position among the
Acanthaceae, we do not find enough
evidence to conclude that Avicennia is more
closely related to Thunbergioideae than to
other Acanthaceae.
6
ACKNOWLEDGEMENTS
Above all I want to thank my supervisor
Jürg Schönenberger for guiding me through
my work and for always having time to help
me. I also thank all my colleagues at Botan
for supporting and helping me in different
ways.
LITERATURE CITED
Benoist, R. 1944. Contribution à la connaissance des
Acanthacées africaines et malgaches. Notul. Syst.
11: 137-151.
Bentham, G. 1876. Acanthaceae. Pp. 1060--1122 in:
Bentham, G. & Hooker, J.D. (eds.), Genera
plantarum, vol. 2. Reeve, London.
Bremekamp, C.E.B. 1955. The Thunbergia species
of the Malesian area. Verh. Kon. Akad.
Wetensch., Afd. Natuurk., Sect. 2. part 50(4): 1--
90.
Bremer, B., Bremer, K., Heidari, N., Erixon, P.,
Olmstead, R.G., Anderberg, A.A., Källersjö,
M. & Barkhordarian, E. 2002. Phylogenetics of
asterids based on 3 coding and 3 non-coding
chloroplast DNA markers and the utility of non-
coding DNA at higher taxonomic levels. Molec.
Pylogen. Evol. 24: 274--301.
Carlquist, S. 1992. Wood anatomy of sympetalous
dicotyledon families: A summary, with
comments on systematic relationships and
evolution of the woody habit. Ann. Missouri. Bot.
Gard. 79 (1): 303-332.
Dahlgren, R. 1975. A system of classification of the
angiosperms to be used to demonstrate the
distribution of characters. Bot. Not. 128: 119-
147.
Duke, N.C., Benzie, J.A.H., Goodall, J.A. &
Ballment, E.R. 1998. Genetic structure and
evolution of species in the mangrove genus
Avicennia (Avicenniaceae) in the Indo-West
Pacific. Evolution 52(6): 1612-1626.
Erdtman, G. 1945. Pollen morphology and plant
taxonomy. IV. Labiatae, Verbenaceae and
Avicenniaceae. Svensk. Bot. Tidskr. 39: 279-285.
Hedrén, M., Chase, M.W. & Olmstead, R.G. 1995.
Relationships in the Acanthaceae and related
families as suggested by cladistic analysis of
rbcL nucleotide sequences. Pl. Syst. Evol. 194:
93--109.
Junell, S. 1934. Zur Gynäceummorphologie und
Systematik der Verbenaceen und Labiaten. Symb.
Bot. Upsal. 4: 1-219.
 7

Lindau, G. 1893. Übersicht über die bisher Scotland, R. W., & Vollesen, K. 2000. Classification
bekannten Arten der Gattung Thunbergia L. f. of Acanthaceae. Kew Bulletin 55: 513-589.
Bot. Jahrb. Syst. 41: 31--41. van Tieghem, M.P. 1898. Avicenniacées et
Maheshwari, P. & Kapil, R.N. 1966. Some indian Symphorémacées: Place de ses deux nouvelles
contributions to the embryology of angiosperms. familles dans la classification. J. Bot. (Morot) 12:
Phytomorphology 16: 239-291. 345-365.
McDade, L.A., & Moody, M.L. 1999. Phylogenetic Tomlinson, P.B. 1995. The botany of mangroves.
relationships among Acanthaceae: Evidence from Cambridge University Press, Cambridge.
noncoding trnL-trnF chloroplast DNA sequences. Zamski, E. 1979. The mode of secondary growth and
Amer. J. Bot. 86: 70--80. the three-dimensional structure of phloem in
McDade, L.A., Masta, S.E., Moody, M.L. & Avicennia. Bot. Gaz. 140(1): 67-76.
Waters, E. 2000. Phylogenetic relationships
among Acanthaceae: Evidence from two
genomes. Syst. Bot. 25: 106-121.
Mohan Ram, H.Y. & Wadhi, M. 1965. Embryology
and the delimitation of the Acanthaceae.
Phytomorphology 15 (1-4): 201-205.
Moldenke, H.N. 1960. Materials toward a
monograph of the genus Avicennia. Phytologia 7
(3-5): 123-168, 179-232, 259-293.
Padmanabhan, D. 1960. The embryology of
Avicennia officinalis. 1. Floral morphology and
gametophytes. Proc. Indian Acad. Sci. Pl. Sci.
B52: 131-145.
Padmanabhan, D. 1964. The embryology of
Avicennia officinalis. 2. Endosperm.
Phytomorphology 14: 442-451.
Profice, S.R. 1988. Mendoncia Vell. ex Vand.
(Acanthaceae): Espécies ocorrentes no Brasil.
Arq. Jard. Bot. Rio J. 29: 201--279.
Sanders, R. W. 1997. The Avicenniaceae in the
Southeastern United States. Harvard Pap. Bot.
10: 81-92.
Saxena, M.R. 1981. Contribution to the
palynotaxonomy of Avicenniaceae Endl. J.
Indian Bot. Soc. 60: 28-31.
Schönenberger, J. & Endress, P.K. 1998. Structure
and development of the flowers in Mendoncia,
Pseudocalyx, and Thunbergia (Acanthaceae) and
their systematic implications. Int. J. Pl. Sci. 159:
446-465.
Schönenberger, J. 1999. Floral structure,
development and diversity in Thunbergia
(Acanthaceae). Bot. J. Linn. Soc. 130: 1-36.
Schwarzbach, A. E., & McDade, L. A. 2002.
Phylogenetic relationships of the mangrove
family Avicenniaceae based on chloroplast and
nuclear ribosomal DNA sequences. Systematic
Botany 27: 84-98.
 8

Molecular phylogenetics and morphological evolution of Thunbergioideae

(Acanthaceae)

Agneta Julia Borg1, Lucinda A. McDade2 & Jürg Schönenberger1

1
Department of Botany, Stockholm University, Lilla Frescativägen 5, 106 91 Stockholm, Sweden,
borg@botan.su.se (author for correspondence).
2
Rancho Santa Ana Botanic Garden, 1500 North College Avenue, Claremont, California 91711-
3157, U.S.A.

Based on nucleotide sequences from three chloroplast DNA regions (rps16, rpl16, trnT-trnL), we infer
detailed phylogenetic relationships within the subfamily Thunbergioideae (Acanthaceae) and among
major lineages of the family as a whole. Taxa were sampled to cover the geographic distribution of
Thunbergioideae and to include all subgenera of the largest genus Thunbergia. All other major lineages of
Acanthaceae were sampled to test monophyly and intrafamilial position of Thunbergioideae. Both
parsimony and Bayesian analyses support Thunbergioideae as monophyletic. The mangrove genus
Avicennia is consistently placed as sister to Thunbergioideae although with only moderate support.
Thunbergia and Mendoncia are both monophyletic in all analyses, and Mendoncia is placed as sister to
Thunbergia plus Pseudocalyx. Relationships within the two largest genera Mendoncia and Thunbergia are
highly resolved and most branches are strongly supported. Our results suggest that the existing
morphology-based classification of Thunbergia partially holds, but needs revision. Based on the
phylogenetic relationships we found it likely that a twining habit is ancestral for the genus Thunbergia.
The thecal awns, characteristic for many species in the genus, have probably evolved from unicellular
bristles. Longitudinal dehiscence over the whole length of the thecae, which is present in many
Thunbergia species, most likely evolved from short slits or pores as present in Pseudocalyx and
Mendoncia.

INTRODUCTION Madagascar, Asia, and Australia. The second

Acanthaceae are a large family of mainly
tropical and subtropical species within the
asterid order Lamiales. The delimitation of
the family has been controversial due to
morphological differences between the
subfamily Acanthoideae (Acanthaceae sensu
stricto), containing the vast majority of all
species in the family, and the two other
subfamilies, the Nelsonioideae and the
Thunbergioideae sensu Scotland & Vollesen
(2000).
The Thunbergioideae comprise five
genera, the largest of which, Thunbergia
Retz., contains about 100 species restricted
to tropical and subtropical regions of Africa,
largest genus, Mendoncia Vell. ex Vand. (ca.
60 species), is most diverse in Central and
South America with some African
representatives and a few species native to
Madagascar. The remaining genera are
Pseudocalyx Radlk. with ca. 7 species in
Africa and Madagascar, and monotypic
Anomacanthus R.D. Good in tropical Africa,
and monotypic Meyenia Nees in India. The
subfamily is characterized by a
predominantly twining habit, enlarged
bracteoles, and a reduced calyx. Further-
more, Thunbergioideae (together with
Nelsonioideae) lack the retinaculate fruits
found in all Acanthaceae s.s., instead
possessing either dry and dehiscent capsules

without retinacula (Thunbergia, Pseudocalyx
and Meyenia) or fleshy drupes (Mendoncia
and Anomacanthus). While there are no
existing subgeneric classifications for
Mendoncia or Pseudocalyx (but see Profice,
1988, for Mendoncia in Brazil), more
attention has been given to Thunbergia.
Lindau (1893) subdivided Thunbergia into
four sections based on morphology and
arrangement of flowers. Bremekamp (1955)
revised and extended Lindau’s subdivision
and proposed eight subgenera. His
classification largely concurs with a recent
study of floral development and structure in
Thunbergia by Schönenberger (1999), but
has never been compared to a hypothesis of
phylogenetic relationships based on DNA
sequence data.
The Thunbergioideae have traditionally
been associated with Acanthaceae (Table 1),
but their markedly different morphology has
led some authors to place them in a separate
family Thunbergiaceae together with the
Nelsonioideae (van Tieghem, 1908a) or even
in two distinct families Thunbergiaceae and
Mendonciaceae (Bremekamp, 1953; Dahl-
gren, 1980; Cronquist, 1981). Other authors,
however, considered the morphological
differences to be insufficient to separate
Thunbergioideae from Acanthaceae and
instead kept them within the family, either as
a tribe (Bentham, 1876), as a subfamily
(Brummitt, 1989; Takhtajan, 1997; Scotland
& Vollesen, 2000), or as two separate
subfamilies Thunbergioideae and Mendonci-
oideae (Lindau, 1895; Thorne, 1992).
Since molecular methods became widely
used, a number of studies have shown that
Thunbergioideae and Acanthaceae s.s. are
closely related, placing Thunbergioideae
either as sister to Acanthaceae s.s. or in a
polytomy within or near Acanthaceae s.s.
(Hedrén & al., 1995; Scotland & al., 1995;
McDade & Moody, 1999). Although based
on a minimal taxon sampling, these same
studies also indicated that Thunbergioideae
(sensu lato) form a natural group. This last
9
was also supported in a study of floral
development and structure by Schönenberger
& Endress (1998). Nelsonioideae were
mostly resolved as sister to all other
Acanthaceae (e.g., Scotland & al., 1995;
McDade & al., 2000). In a review of
morphological and molecular studies in
Acanthaceae, Scotland & Vollesen (2000)
presented a classification of the family in the
broad sense, including both Thunbergioideae
and Nelsonioideae. More recently and quite
surprisingly, a molecular study by
Schwarzbach & McDade (2002) implied that
the mangrove genus Avicennia L., usually
treated as a separate family in Lamiales or as
a genus within Verbenaceae, is also part of
Acanthaceae. In their study, Avicennia is
consistently placed as sister group to
Thunbergioideae albeit with weak support.
Although the delimitation of the
Acanthaceae now seems well supported,
exact relationships among Acanthoideae,
Thunbergioideae, Nelsonioideae and
Avicennia remain unclear. Furthermore,
despite the number of morphological and
molecular studies showing that Thunberg-
ioideae belong in Acanthaceae, no molecular
study has so far included more than a couple
of representatives from Thunbergia and
Mendoncia. Accordingly, phylogenetic
relationships within Thunbergioideae are
currently not well understood.
The main goals of the present study are
1) to test whether Thunbergioideae are
monophyletic, 2) to determine the exact
position of the Thunbergioideae among the
other acanthaceous lineages, 3) to elucidate
evolutionary relationships within Thun-
bergioideae, 4) to find out whether
molecular evidence is congruent with earlier,
morphology-based attempts to classify
Thunbergioideae as a whole and the genus
Thunbergia in particular (e.g., Lindau, 1895;
Bremekamp, 1955), and 5) to discuss the
evolution of a number of morphological
traits traditionally used for classification of
the group.

Table 1. Classification of Acanthaceae.
Lindau 1895 Bremekamp 1953
Acanthoideae Acanthaceae (s.s.)
Thunbergioideae Thunbergiaceae
Mendoncioideae Mendonciaceae
Nelsonioideae Nelsonioideae referred to
Scrophulariaceae
MATERIALS AND METHODS
Taxon sampling
We sampled molecular characters from
30 species from the three major genera of
Thunbergioideae (Thunbergia, Mendoncia,
and Pseudocalyx). The sampled Thunbergia
species (21 species) represent all 8
subgenera circumscribed by Bremekamp
(1955) and also cover the geographic range
of the genus. The sampling also includes T.
arnhemica, the only Thunbergia species
native to Australia. Our sampling
furthermore covers the main distribution
areas of the genus Mendoncia (8 species)
with representatives from Tropical West
Africa, Madagascar, Central and South
America. The small genus Pseudocalyx is
represented by a single African/Malagasy
species.
Sampling of the other acanthaceous
lineages include two species from each of
the two tribes of the Acanthoideae; the
Acantheae and Ruellieae (sensu Scotland &
Vollesen 2000), single representatives of
three out of six genera of Nelsonioideae, and
three out of 8 species of Avicennia (sensu
Tomlinson, 1995). As an out-group we used
the genus Schlegelia Miq., which is possibly
sister to Acanthaceae (71% jackknife
support) as shown in a molecular study of
the asterids by Bremer & al. (2002).
Molecular methods
Total genomic DNA was extracted from
leaf material dried in silica gel or from
recently collected herbarium specimens,
10
Scotland & Vollesen 2000
Acanthoideae
Thunbergioideae (s.l.)
Nelsonioideae
either using DNeasy kits (Qiagen), or
following the CTAB protocol by Doyle and
Dickson (1987). CTAB samples were
cleaned with QIAquick PCR Purification Kit
(Qiagen). Three chloroplast (cp) DNA
regions, the rps16 intron, the rpl16 intron,
and the trnT-trnL intergenic spacer, were
amplified and sequenced for all taxa. The
rps16 intron is a widely used cp DNA
region, which has been shown to be
informative also among Acanthaceae
(McDade & al., 2005). The rps16 intron was
amplified using the primers of McDade & al.
(2005). To amplify the rpl16 intron, primers
F71 (Jordan & al., 1996) and R1516 by
Baum & al. (1998) were used. The trnT-trnL
intergenic spacer was amplified using the
trnA2 primer of Cronn & al. (2002) and the
b primer of Taberlet & al. (1991).
Polymerase chain reaction (PCR)
amplifications for all three regions used the
following thermal cycling protocol:
preheating at 94ºC for 2 min 30 s followed
by 35 cycles of denaturation at 94ºC for 45 s,
annealing at 52ºC for 1 min, and extension at
74ºC for 1 min 20 s. A final 10 min
extension at 72ºC was followed by cooling
to 4ºC. Amplified PCR products were
purified by vacuum filtration using
MultiScreen Vacuum Manifold (Millipore).
The same primer pairs were used for the
sequencing reactions together with the Big
Dye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems). Sequencing was
accomplished on an automated capillary ABI
3100 Genetic Analyzer (Applied
Biosystems). Both strands of the three

regions were sequenced for all taxa to verify
the complementary strands against each
other. All sequences were proofread and
double-checked against electropherograms
and then assembled using the Staden
Software Package version 1.6.0
(http://staden.sourceforge.net/).
Alignment and analysis
Sequences were aligned by eye after an
initial alignment was created with Clustal W
(Thompson & al., 1994) in BioEdit version
7.0.1 (Hall, 1999). A region near the middle
of the rpl16 intron was extremely variable in
length (1 to 235 base pairs) and we were
unable to align the sequences with
confidence. This region was therefore
omitted from all further analyses.
Phylogenetic reconstruction
Phylogenetic analyses were performed
using maximum parsimony (MP) as well as
Bayesian inference of phylogeny. MP
analyses were carried out in PAUP version
4.0b10 (Swofford, 2002). All characters and
character states were weighted equally and
gaps were treated as missing characters.
Four data sets were analyzed; one for each
cp DNA region and one combined data set
including all three regions. The four matrices
were analyzed by employing a heuristic
search strategy with 10,000 replicates of
random taxon addition, holding 100 trees at
each step during stepwise addition, using the
tree-bisection-reconnection branch-swapping
algorithm, saving multiple equally
parsimonious trees, and with the steepest
descent option in effect. The resulting MP
trees from each analysis were used to
produce strict consensus trees. Relative
branch support was measured by non-
parametric bootstrap analysis (Felsenstein,
1985) using a heuristic search strategy,
10,000 bootstrap replicates with 10 random
sequence additions and holding 1 tree at
each step during stepwise addition.
11
Bayesian analyses (BA) were carried out
using MrBayes version 3.1.2 (Huelsenbeck
& Ronquist, 2001; Ronquist & Huelsenbeck,
2003). The software program MrAIC version
1.4.2 (Nylander, 2004) was used to compare
24 nucleotide substitution models based on
the Akaike and Bayesian information criteria
(BIC). We let the program evaluate models
for the three separate data partitions as well
as for the combined data set. The same
models were chosen with the same ranking
for all data sets and we therefore proceeded
with the combined data set only. The two
best fitting models were applied to the data
in separate analyses, and each analysis was
carried out in MrBayes as follows: Two
parallel runs (default in MrBayes version
3.1.2), each using one cold and three heated
chains, were run for 5,000,000 generations
with sample trees saved every 100
generations. The first 12,500 (25%) of the
sample trees from each run were discarded
(burn in), and a maximum a posteriori tree
was constructed by summarizing the
remaining 75,000 trees from parallel runs in
a majority rule consensus tree, thus yielding
the posterior probability (PP) values for each
clade.
RESULTS
Table 2 presents data set characteristics
for the three cp regions and the combined
data set. The combined data set included
2899 aligned positions, of which 1063 were
variable and 597 were parsimony
informative. The trnT-trnL intergenic spacer
provided the highest percentage of
parsimony informative characters (22.1 %),
followed by the rpl16 intron (21.4 %) and
the rps16 intron (18.2 %).
Parsimony analyses
MP analyses of the three separate cp
regions yielded congruent results and the
strict consensus trees did not present any

Table 2. Description of data sets and trees resulting from maximum parsimony analyses (excluding uninformative
characters).
Characteristic rps16 intron
Aligned length 959
Variable positions (%) 316 (33.0)
Parsimony informative (%) 175 (18.2)
Consistency index 0.7143
Retention index 0.8801
Number of shortest trees 184
Length of shortest tree 478
topological conflicts (trees not shown here,
see Table 2). The combined MP analysis
resulted in eight most parsimonious trees,
summed up in a strict consensus tree (Fig.
1A). Combining the three data matrices
improved overall tree resolution and branch
support, but the combined analysis did not
find any clades not present in at least one of
the strict consensus trees of the individual
data sets.
The deepest split in the phylogeny is
between a monophyletic Nelsonioideae
(BS=100) and the remaining in-group taxa
(BS=89). Acanthoideae, Thunbergioideae
and Avicennia are all well supported
monophyletic groups (BS=97, 100 and 100,
respectively). Avicennia is moderately
supported as sister to Thunbergioideae
(BS=68) and Acanthoideae are sister to the
two latter taxa. The MP analysis fully
supports the monophyly of Thunbergioideae
as a whole and clearly shows that
Thunbergia as well as Mendoncia are
monophyletic (both BS=100). Pseudocalyx
is consistently resolved as sister to
Thunbergia (BS=99) and Mendoncia is in
turn sister to these two genera.
Bayesian analyses
The same models were preferred using
the Akaike information criterion (AIC) and
Bayesian information criterion (BIC). Two
models fitted the data (together correspond-
ing to a cumulative Akaike weight of 1.000).
12
rpl16 intron trnT-trnL spacer combined
987 953 2899
369 (37.4) 378 (39.7) 1063 (36.7
211 (21.4) 211 (22.1) 597 (20.6
0.6506 0.7088 0.6816
0.8285 0.8813 0.8608
162 1166 8
615 578 1675
The best scored model for all four data sets
was a general time-reversible model with
gamma-distributed rates (GTR+Γ). The
second best model for all data sets was a
GTR model with gamma-distributed rate and
a proportion of invariant sites (GTR+Γ+I).
Since the same model ranking was proposed
for each of the three individual data sets as
well as the combined data set, we decided to
analyze the three data matrices as a single
partition.
The Bayesian analysis provided a tree
topology almost identical to the MP tree
(Fig. 1B and Fig. 2), the difference being
that the BA majority rule tree is somewhat
better resolved than the MP strict consensus
tree. The PP values are generally high. The
Bayesian analysis found four branches that
are not present in the MP tree: 1) The basal
trichotomy in Thunbergia is resolved,
suggesting that the clade containing T.
erecta, T. affinis and T. guerkeana (PP=1) is
sister to all other Thunbergia species
(PP=0.76); 2) T. coccinea and T. grandiflora
are more closely related to each other
(PP=0.83) than to T. laurifolia; 3) T.
capensis and T. pondoensis are more closely
related to each other (PP=0.98) than to T.
atriplicifolia, and 4) together these three
species constitute the sister group to T.
dregeana and T. galpinii (PP=0.99) while T.
angulata and T. convolvulifolia have
diverged earlier on.
 13

Fig. 1. Phylogenetic trees based on analyses of the combined data set. A, Strict consensus of eight shortest trees
resulting from maximum parsimony (MP) analysis; numbers above branches are bootstrap values. B, Majority rule
consensus tree resulting from Bayesian analysis; numbers above branches are Bayesian posterior probabilities;
asterisks indicate nodes not present in MP tree.

DISCUSSION genera show all general characteristics of

Monophyly and subdivision of Thunberg-
ioideae
Our results suggest that Thunbergioideae
are monophyletic (BS=100; PP=1.0).
Uncertainties remain regarding the two
monotypic genera Anomacanthus and
Meyenia, of which we were unable to get
material. However, the inclusion of these
species is unlikely to change the
circumscription of the subfamily as both
Thunbergioideae (Good, 1923; Brummitt,
1989), and the single Meyenia species was
originally described as belonging to
Thunbergia by Wallich (1826; see also
Matthew, 1983). Nevertheless, we aim to
explore the exact positions of Anomacanthus
and Meyenia in future studies as they may
have bearing on the delimitation of the larger
genera.
The monophyly of Thunbergioideae is
also supported by morphological
 14

Fig. 2. Branch lengths and geographical distribution. Phylogram resulting from Bayesian analysis of the combined
data set. Branch lengths are proportional to number of changes. Abbreviations: SAfr, South Africa; Mad,
Madagascar; TAfr, Tropical Africa; Aus, Australia; TAm, Tropical America; PanT, Pantropical; WW, Worldwide.

characters. The subfamily is characterized by endothecium, and similar inflorescences

having flowers subtended by two large
persistent bracteoles (Fig. 4A-D), strongly
reduced calyces (Fig. 4E-H), and a tendency
to twine. Other characters connecting
Thunbergia, Mendoncia and Pseudocalyx
are lignified unicellular bristles on the
anthers (Fig. 3, Fig. 4I), an ephemeral
(Schönenberger & Endress, 1998).
Based on the present taxon sampling,
Thunbergia and Mendoncia are two
monophyletic genera and all analyses
suggest that Thunbergia and Pseudocalyx
are sister groups (BS=99; PP=1.0), and
together they are sister to Mendoncia. There

are several morphological traits that
demonstrate the close relationship between
Thunbergia and Pseudocalyx. The most
obvious one is perhaps that Thunbergia and
Pseudocalyx have dry capsules, like all other
Acanthaceae, whereas Mendoncia possesses
fleshy drupes; an exceptional character in
Acanthaceae only shared with Anom-
acanthus. Another difference in the fruit is
that Thunbergia and Pseudocalyx have two
fertile locules, while in Mendoncia only one
of two initiated locules develops fully
(Schönenberger & Endress, 1998).
The dissimilarities prompted Lindau
(1895) to place Mendoncia in a separate
subfamily Mendoncioideae (Table 1).
Bremekamp (1953) went even further and
raised Lindau’s Mendoncioideae to family
rank, mainly due to the divergent fruit
characters. However, several authors have
pointed out characters linking Mendoncia to
Thunbergioideae s.s. and in particular to
Pseudocalyx, which shares several features
with Mendoncia including stigma shape and
anther dehiscence (Fig. 3), indumentum
(Brummitt, 1989; Schönenberger & Endress,
1998), and relative size of corolla lobes
(Radlkofer, 1883). Brummitt (1989) noted
that Pseudocalyx has important characters in
common with both Thunbergia and
Mendoncia, and considered it impossible to
place Mendoncia in a separate family. Later,
Schönenberger & Endress (1998) confirmed
this and showed that the drupaceous fruits in
Mendoncia and the dry capsules in
Thunbergia and Pseudocalyx actually have
an identical bauplan and that differences
arise late during floral development. The
main differences between Thunbergia and
Pseudocalyx were thought to lay in bracteole
characteristics, stigma structure, and anther
dehiscence (i.e. longitudinal vs. porate;
Bremekamp, 1955). However, Schönen-
berger & Endress (1998) showed that the
differences in dehiscence mode are not clear
cut between the two genera as intermediate
dehiscence patterns occur in both groups.
15
Subgeneric relationships in Mendoncia and
Thunbergia
There is no comprehensive subgeneric
taxonomic treatment of Mendoncia, although
some of the species have earlier been
ascribed to two isolated genera. Three of the
Mendoncia species in the present study, M.
flagellaris, M. cowanii and M.
phytocrenoides, have earlier been ascribed to
Monachochlamys Baker (M. flagellaris by
Baker 1883; M. cowanii and M.
phytocrenoides by Moore, 1929) and the
latter two have also been ascribed to
Afromendoncia Gilg (A. phytocrenoides by
Gilg in Lindau, 1893; A. cowanii by Moore,
1906). Both genera were sunk back into
Mendoncia by Benoist (1944), who argued
that the few existing divergent characters in
Monachochlamys are insufficient to
distinguish them as a genus, and that some
of the observations underlying the separation
of Afromendoncia were actually incorrect.
Our data show that neither Monachochlamys
nor Afromendoncia are monophyletic.
However, relationships among Mendoncia
species correspond well to the geographic
distribution of the genus (Fig. 3). The genus
displays a trans-Atlantic disjunction, and the
present results support a clade consisting
entirely of American species, while M.
phytocrenoides, which is sister to the
American group, occurs in tropical regions
of the African continent and the sister group
to these two lineages is endemic to
Madagascar.
Bremekamp (1955) presented a sub-
generic classification of Thunbergia, with
eight subgenera determined mainly by
stigma and anther morphology, leaf and
inflorescence features as well as growth
form. A study of structure and development
of flowers in Thunbergia (Schönenberger,
1999) largely agrees with this classification.
The present molecular phylogenetic study
partly support Bremekamp’s subgenera.
Based on the present taxon sampling, the
subgenera Macrosiphon and Coniostephanus
 16

Fig. 3. Distribution of morphological characters in Thunbergioideae on the majority rule consensus tree resulting
from Bayesian analysis of the combined data set, .Bremekamp’s (1955) subgeneric classification of Thunbergia is
given on the right and geographic ranges are indicated after taxon names. Abbreviations: SAfr, South Africa; Mad,
Madagascar; TAfr, Tropical Africa; Aus, Australia; TAm, Tropical America. Habit: creeping, twining, erect. Calyx
type: Many-lobed, truncate, irregularly truncate, five-lobed, six-lobed. Anther appendages: none, multicellular awns,
unicellular bristles. Anther dehiscence: Long slits, short slits in lower half of theca, short slits in upper half of theca,
pores. Stigma type (Schönenberger 1999): adaxial lobe folded and abaxial spreading, funnel-shaped and both lobes
equal, funnel-shaped and adaxial lobe more folded than abaxial lobe, funnel-shaped with short lobes and trichome
tufts, both lobes equal and folded, both lobes very short. Data from Barker, 1986; Braz & al., 2002; Bremekamp,
1955; Breteler, 1998; Clarke, 1912; Heine, 1966; Retief & Reyneke, 1984; Schönenberger 1999; Turrill, 1919; A.J.
Borg personal observations.

constitute a monophyletic group (clade I in
Fig. 3) of African plants with a 10-15
toothed calyx (Fig. 4E) and unicellular
lignified bristles at the base of the thecae
(Fig. 4I). Furthermore, the thecae open by
longitudinal slits that are restricted to the
upper half, a characteristic not found
elsewhere among examined species of
Thunbergia (Fig. 4I). What separates the two
subgenera is that in Coniostephanus, the

upper stigmatic lobe is folded and the lower
is flat and spreading, whereas in
Macrosiphon the two stigmatic lobes are
more or less equal in shape (Fig. 4M-N).
Bremekamp (1955) commented that
Macrosiphon and Coniostephanus must be
closely related, and our molecular data
strongly support them as sister groups
(BS=100; PP=1.0). Moreover, Bayesian
analyses suggest that the two subgenera are
sister to the clade containing all other
Thunbergia species.
Subgenus Hypenophora is represented in
our study by T. togoensis and T.
battiscombei. According to our results they
are indeed closely related, but T. petersiana,
referred by Bremekamp (1955) to subgenus
Parahexacentris, is sister to T. togoensis.
This classification by Bremekamp most
likely is a mistake, since the species would
be placed in Hypenophora by the characters
he used for his classification. The clade of
Hypenophora and T. petersiana (clade II in
Fig. 3) is rather distinct: all are erect herbs,
have a basically 6-lobed calyx (Fig 4F),
anthers that open by short slits in the lower
half of the thecae, and anthers provided with
curved awns (Fig. 4J). In addition, as
Bremekamp (1955) noted, a remarkable
feature of Hypenophora (and T. petersiana)
is the funnel shaped stigma with a tuft of
trichomes emerging on each side (Fig. 4O).
This combination of characters is not present
in any other Thunbergia clade and the floral
morphology of this clade stands out against
the rest of the genus. The distinctiveness of
the clade is also apparent at the molecular
level as the branch in the phylogeny leading
to this clade is particularly long, i.e., has
accumulated a relatively large amount of
nucleotide substitutions (Fig. 2).
Subgenus Thamnidium consists of only
two species, represented here by T. kirkii,
which is characterized by having a distinctly
pentamerous calyx and stigmas without
lateral tufts, as opposed to its closest
relatives in the subgenus Hypenophora
17
(including T. petersiana). Also, Thamnidium
have thecae that open by long slits extending
over the whole length of the thecae, and in
this respect is more similar to most other
Thunbergia species than they are to
subgenus Hypenophora. Thamnidium and
Hypenophora, including T. petersiana, occur
in tropical regions in Africa and form a well
supported clade (III) in the present analysis
(BS=99; PP=1.0).
Subgenus Hexacentris (clade IV) is a
rather well defined group of Asian woody
climbers with irregular, more or less truncate
calyces (Fig. 4G-H), conspicuously awned
thecae opening by long slits (Fig. 4K), and
funnel shaped stigmas with short lobes (Fig.
4P). Hexacentris has earlier been proposed
by some authors to constitute a separate
genus (Nees von Esenbeck, 1847; van
Tieghem, 1908b), and the group is also
strongly supported as being monophyletic by
our data (BS=100; PP=1.0). Bremekamp
(1955), however, found no reason to raise
Hexacentris to genus level, and molecular
data also show that it is deeply nested within
Thunbergia.
Hexacentris is sister to a large clade (V)
consisting of species which also have thecae
that open by long slits, but they differ from
Hexacentris by having calyces with 10-15
lobes (Fig. 4E) Bremekamp (1955) referred
the species in this clade to three subgenera:
Parahexacentris, Eu-thunbergia and
Adelphia. Parahexacentris is the largest
subgenus within Thunbergia containing
African and Malagasy species. It is
characterized by two well developed stigma
lobes (Fig. 4M) in combination with rather
long thecal awns (Fig. 4K). The same type of
stigma is present in the small subgenus Eu-
thunbergia. The thecal bases, however, are
blunt in Eu-thunbergia (Fig. 4L). Molecular
data reveal that Eu-thunbergia, represented
by T. capensis, is in fact nested within
Parahexacentris. It is worth mentioning,
however, that there is at least one other
species in Parahexacentris which also lacks
 18

Fig. 4. Floral morphology. A-D, Flowers of Thunbergioideae. Scale bars = 1 cm: A, Thunbergia convolvulifolia; B,
T. petersiana; C, Pseudocalyx saccatus; D, Mendoncia retusa. E-H, Calyx types in Thunbergia. Scale bars = 1 mm:
E, many-lobed (T. convolvulifolia, corolla removed); F, six-lobed (T. petersiana, corolla removed); G, irregularly
truncate (T. coccinea, corolla removed); H, truncate (T. laurifolia). I-L, Anther appendages and dehiscence in
Thunbergia. Scale bars = 1 mm: I, multicellular bristles and short slits in upper half (T. erecta); J, multicellular awns
and short slits in lower half (T. togoensis); K, multicellular awns (indicated by arrowheads) and long slits (T.
angulata); L, anther appendages absent and long slits (T. fragrans). M-P, Stigma types in Thunbergia. Scale bars =
0.5 mm: M, adaxial lobe folded and abaxial spreading (T. convolvulifolia); N, both lobes equal and folded (T.
guerkeana); O, funnel-shaped with short lobes and trichome tufts (indicated by arrowhead, T. petersiana); P, funnel-
shaped and adaxial lobe more folded than abaxial lobe (T. coccinea).

thecal appendages, i.e., the Malagasy T.
convolvulifolia (pers. obs.), which Breme-
kamp probably was not able to study himself
(he mentions that his classification is
partially based on descriptions; Bremekamp,
1955). The third subgenus, Adelphia,
contains a number of Asian/Australian
species, represented by T. arnhemica and T.
fragrans in the present analyses. The clade
formed by these two species (VI) is also
nested within subgenus Parahexacentris.
Adelphia has a different kind of stigma
morphology with funnel shaped stigmata and
equally sized lobes and thecae usually
lacking appendages (Bremekamp, 1964).
Flowers of this subgenus exhibit a
hawkmoth pollination syndrome with only
weakly monosymmetric flowers, a white
corolla, and narrow corolla throat and tube
(Schönenberger, 1999). The species included
by Bremekamp in Adelphia have often been
regarded as varieties of T. fragrans
(Bremekamp, 1955), but whereas T. fragrans
originates from India and Sri Lanka, T.
arnhemica is only known from northern
Australia, and is the only Thunbergia native
to Australia (Bremekamp, 1964; Barker,
1986). At the molecular level, differences in
nucleotide sequences between T. fragrans
and T. arnhemica are as or more numerous
than between many other species pairs in
Thunbergia (Fig. 2).
Thus, Bremekamp’s (1955) classification
partially stands, but the large subgenus
Parahexacentris is clearly paraphyletic with
both Eu-thunbergia and Adelphia nested
within it. Instead, a number of other
monophyletic groups involving representa-
tives of Bremekamp’s subgenus Parahexa-
centris can be distinguished based on our
analysis: A South African clade (clade VII in
Fig. 3) comprising Eu-thunbergia and
Parahexacentris p.p., all with a creeping
habit (Retief & Reyneke, 1984) finds strong
support in the Bayesian analysis (PP=0.99).
Together with two twining Malagasy
representatives of Parahexacentris, i.e., T.
19
convolvulifolia and T. angulata, the latter
species form a strongly supported clade
(clade VIII; BS=100, PP=1.0). The clade
(BS=91, PP=100) with the two remaining
species of Bremekamp’s Parahexacentris,
i.e. T. alata and T. gregorii, is sister to
subgenus Adelphia.
Habit, calyx morphology, and anther
dehiscence patterns seem to reflect
molecular evolutionary relationships part-
icularly well among Thunbergia species
(Fig. 3), whereas the presence or absence of
anther appendages and stigma structure are
somewhat more labile and most likely are
closely correlated with differences in
pollination biology (see Schönenberger,
1999, for discussion).
The position of Thunbergioideae among the
other acanthaceous lineages
The results agree with the intrafamilial
position of Thunbergioideae proposed in
earlier higher-level studies (e.g., Hedrén &
al., 1995; Scotland & al., 1995; McDade &
al., 2000), with generally high bootstrap
support values and Bayesian posterior
probabilities. According to our analyses, the
closest relative of Thunbergioideae is the
pantropical mangrove genus Avicennia. This
relationship is supported by high Bayesian
PP values (0.98) and moderate bootstrap
support (BS=68). Schwarzbach & McDade
(2002) showed that Avicennia belong in
Acanthaceae and they too found consistent
molecular evidence for a sister relationship
between Thunbergioideae and Avicennia
(max. BS=78) using parsimony and
maximum likelihood analyses of chloroplast
and nuclear DNA regions. They also
considered morphological traits but these did
not provide evidence for a closer relationship
of Avicennia to Thunbergioideae than to any
other acanthaceous lineage. Avicennia
appears to be morphologically highly
specialized due to adaptations to the
mangrove habitat, and exhibit many
convergent characters found in several

mangrove taxa (Sanders, 1997; Schwarzbach
& McDade, 2002). Taxonomic adjustments
concerning Avicennia will have to await
future studies, both at the molecular and
morphological level.
Acanthoideae are sister to Thunbergi-
oideae plus Avicennia with strong support
(PP=1.00; BS=89). This was also found by
Schwarzbach & McDade (2002). Acantho-
ideae are distributed from the tropics to
temperate regions and are recognized by
their unique fruit type, an explosively
dehiscent capsule with retinacula functioning
as a lever ejecting the seeds from the fruits.
Our analyses place the subfamily
Nelsonioideae, here represented by one
species from each of its two major genera
(Staurogyne and Elytraria) plus one smaller
genus (Anisosepalum), as sister to the clade
comprising all other Acanthaceae (BS=89,
PP=1.0). These findings are consistent with
previous molecular studies (e.g., Scotland &
al., 1995; McDade & al., 2000). Based on
our limited taxon sampling, the Nelsoni-
oideae appear monophyletic (BS=100,
PP=1.0). However, Scotland & Vollesen
(2000) were unable to find unambiguous
morphological characters supporting the
monophyly of Nelsonioideae, and no
molecular study so far has included all
genera of the subfamily. Nelsonioideae
differ in several important aspects from other
Acanthaceae. Like Thunbergioideae, they
lack the retinaculate fruits characterizing
Acanthoideae. Furthermore, Nelsonioideae
have alternate bracts in the inflorescence and
persistent endosperm as opposed to
decussate bracts and seeds without
endosperm in other Acanthaceae. Like
Acanthoideae, Nelsonioideae are widespread
in tropical and warm temperate regions.
Molecular data has certainly enhanced
the understanding of evolutionary
relationships among the first branching
lineages of Acanthaceae, but some questions
remain. The traditionally conflicting views
regarding the taxonomy and systematic
20
position of Thunbergioideae reflect the fact
that indisputable morphological synapo-
morphies linking Thunbergioideae to other
Acanthaceae have yet to be discovered.
More molecular work as well as detailed
comparative morphological studies are
needed to shed light on structural evolution
of Avicennia, and molecular work on
Nelsonioideae will provide further inform-
ation about the early evolution of Acantha-
ceae. The geographic ranges of genera and
species in the basal lineages of Acanthaceae
(see Fig. 2) raise some questions regarding
the geographic origin of the various taxa.
The disjunction in Mendoncia and a number
of other trans-Atlantic disjunct genera are
listed by Renner (2004), and are suggested to
have occurred by long-distance dispersal or,
in older groups (i.e., higher taxonomic
ranks), may be attributed to the breakup of
continents. The test of these and of more
detailed hypotheses on the biogeographic
history of Acanthaceae and in particular of
Thunbergioideae and its subclades will,
however, have to await future phylogenetic
analyses based on a broader taxon sampling.
ACKNOWLEDGEMENTS
We thank Luis Diego Gomez, Eugenio
González and Francisco Campos from the
Organization for Tropical Studies (OTS) for
help with collection permits in Costa Rica;
Barry Hammel, Nelson Zamora and other
staff at the National Biodiversity Institute
(INBio) and Paul Foster at the Reserva
Ecológica Bijagual for assistance and help in
the field in Costa Rica; Sylvain
Razafimandimbison for organizing a field
trip to Madagascar; and Carrie Kiel and
Anbar Khodabandeh for sharing their
knowledge in the molecular lab. For
providing leaf material, we are grateful to
Alexandre Antonelli, Thomas F. Daniel,
Paul I. Forster and Quentin Luke. Finally,
we thank two anonymous reviewers for

critically reviewing the manuscript.
Financial support for this research was
received from the Jessup-McHenry Fund
(Philadelphia Academy of Natural Sciences)
and Hammarlundfonden (Stockholm
University).
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 23

Appendix. Taxon; Location; Voucher; GenBank accession numbers rps16, rpl16, trnT-trnL.
Anisosepalum alboviolaceum (Benoist) E. Hossain; cultivated; National Botanic Garden of Belgium 95-0025-35
(BR); EU315785, EU315826, EU315867. Avicennia bicolor Standl.; Costa Rica; Borg 10 (S); EU315786,
EU315827, EU315868. Avicennia germinans (L.) Stearn; USA; Schönenberger 725 (voucher in ethanol) (S);
EU315787, EU315828, EU315869. Avicennia marina (Forssk.) Vierh.; Madagascar; Borg 35 (S); EU315788,
EU315829, EU315870. Crossandra strobilifera (Lam.) Benoist; Madagascar; Borg 24 (S); EU315789, EU315830,
EU315871. Ecbolium madagascariense Vollesen; Madagascar; Borg 37 (S); EU315790, EU315831, EU315872.
Elytraria imbricata Pers.; Bolivia; Daniel 10108 (CAS); EU315791, EU315832, EU315873. Gymnostachyum
ceylanicum Arn. & Nees; cultivated; Borg 55 (S); EU315792, EU315833, EU315874. Justicia rizzinii Wassh.;
cultivated; Borg 51 (S); EU315793, EU315834, EU315875. Mendoncia aspera Nees; Bolivia; Schönenberger 416
(Z); EU315794, EU315835, EU315876. Mendoncia cowanii (S. Moore) Benoist; Madagascar; Schönenberger A-49
(UPS); EU315795, EU315836, EU315877. Mendoncia flagellaris Benoist; Madagascar; Borg 47 (S); EU315796,
EU315837, EU315878. Mendoncia glabra Nees; Bolivia; Schönenberger 440 (Z); EU315797, EU315838,
EU315879. Mendoncia lindavii Rusby; Honduras; Daniel 9510 (CAS); EU315798, EU315839, EU315880.
Mendoncia phytocrenoides Benoist; Cameroon; Schönenberger 50 (K); EU315799, EU315840, EU315881.
Mendoncia retusa Turrill; Panama, Daniel 8061 (CAS); EU315800, EU315841, EU315882. Mendoncia velloziana
Mart.; Brazil; Antonelli & Andersson 287 (GB); EU315801, EU315842, EU315883. Pseudocalyx saccatus Radlk.;
Madagascar; Schönenberger A-168 (UPS); EU315802, EU315843, EU315884. Schlegelia fastigiata Schery; Costa
Rica; Borg 4 (S); EU315784, EU315825, EU315866. Staurogyne letestuana Benoist; cultivated; National Botanic
Garden of Belgium 20000119-77 (BR); EU315803, EU315844, EU315885. Thunbergia affinis S. Moore;
cultivated; National Botanic Garden of Belgium 95-0081-92 (BR); EU315804, EU315845, EU315886. Thunbergia
alata Bojer ex Sims; cultivated; Borg 50 (S); EU315805, EU315846, EU315887. Thunbergia angulata Hils. et
Bojer ex Hook.; Madagascar; Borg 26 (S); EU315806, EU315847, EU315888. Thunbergia arnhemica F. Muell.;
Australia; Forster 11819 (BRI); EU315807, EU315848, EU315889. Thunbergia atriplicifolia E. Mey.; South
Africa; Daniel 9350 (CAS); EU315808, EU315849, EU315890. Thunbergia battiscombei Turrill; cultivated; Daniel
s.n. (CAS); EU315809, EU315850, EU315891. Thunbergia capensis Retz.; cultivated; Daniel s.n. (CAS);
EU315810, EU315851, EU315892. Thunbergia coccinea Wall.; cultivated; Schönenberger 144 (Z); EU315811,
EU315852, EU315893. Thunbergia convolvulifolia Baker; Madagascar; Borg 44 (S); EU315812, EU315853,
EU315894. Thunbergia dregeana Nees; South Africa; McDade & Balkwill 1242 (PH); EU315813, EU315854,
EU315895. Thunbergia erecta T. Anderson; cultivated; Borg 49 (S); EU315814, EU315855, EU315896.
Thunbergia fragrans Roxb.; cultivated; Schönenberger 129 (Z); EU315815, EU315856, EU315897. Thunbergia
galpinii Lindau; South Africa; McDade & Balkwill 1250 (PH); EU315816, EU315857, EU315898. Thunbergia
grandiflora Roxb.; cultivated; Daniel s.n. (CAS); EU315817, EU315858, EU315899. Thunbergia gregorii S.
Moore; cultivated; Daniel s.n. (CAS); EU315818, EU315859, EU315900. Thunbergia guerkeana Lindau; Kenya;
Luke & al. 6197 (EA); EU315819, EU315860, EU315901. Thunbergia kirkii Hook. f.; cultivated; Botanic Garden
of the University of Zürich 1988-1164 (Z); EU315820, EU315861, EU315902. Thunbergia laurifolia Lindl.,
cultivated; Schönenberger 16 (Z); EU315821, EU315862, EU315903. Thunbergia petersiana Lindau; cultivated;
Sweden, Schönenberger 147 (UPS); EU315822, EU315863, EU315904. Thunbergia pondoensis Lindau; South
Africa; Daniel 9331 (CAS), EU315823, EU315864, EU315905. Thunbergia togoensis Lindau; cultivated; Royal
Botanic Gardens Kew 1966-50003; EU315824, EU315865, EU315906.
 24

Floral structure and development in Avicennia

Agneta Julia Borg & Jürg Schönenberger

Department of Botany, Stockholm University, Lilla Frescativägen 5, 106 91 Stockholm, Sweden,

borg@botan.su.se (author for correspondence).

Phylogenetic relationships of the small black mangrove genus Avicennia have been difficult to trace due to
the presence of many convergent characters related to the mangrove environment. Recent molecular data
suggest a close relationship to Thunbergioideae, a subfamily within the Acanthaceae (Lamiales), but
morphological support for the new findings have been equivocal. Floral structure and development are
here studied in three species of Avicennia and are discussed in relation to other Acanthaceae and Lamiales
in general. Our developmental studies reveal that the apparently tetramerous flowers of Avicennia have a
basically pentamerous floral ground plan. Additional characters shared with Thunbergioideae include left
contort corolla aestivation, collateral ovule arrangement, thickened filament bases with glandular hairs, the
presence of pollen sack placentoids, unequal stigma lobes and similarities in fruit morphology. In general,
floral morphology and anatomy of Avicennia are rather undistinguished, and none of the features
mentioned above seems to provide a clear synapomorphy for the clade with Avicennia and
Thunbergioideae as most of them are also found in other Acanthaceae, and in some cases even in other
Lamiales. Floral development and floral structure do not contradict a placement of Avicennia within
Acanthaceae, but neither do they provide any clear synapomorphies for the group as a whole and likewise
do they also not provide strong evidence concerning the exact systematic position of Avicennia within the
family.

INTRODUCTION environmental factors (Duke & al., 1998),

The black mangrove, Avicennia, is a
small genus comprising around 8 species
(Tomlinson, 1995) with a wide geographic
distribution along the world’s tropical coasts.
They often make up an important component
of mangrove forests, and like other
mangroves, Avicennia possesses a number of
adaptations to the distinct mangrove habitat.
These include several convergent features
such as specialized erect, lateral roots
developed as pneumatophores, longitudinal
air chambers in the cortex of the
pneumatophores, tolerance to high salt
concentrations, xerophytic leaves with well-
developed hypodermis and viviparous fruits
which are sea-water dispersed. To large
extent morphological characters in Avicennia
appear to be strongly influenced by
making it a difficult task to trace the
evolutionary history of the genus. This is
reflected in the various efforts by earlier
taxonomists to find the closest relatives of
Avicennia, and various suggestions of how
to treat the genus have been put forward
(Table 1). However, while many attributes
are influenced by environmental factors,
some features, particularly of the flowers,
have been found to mirror the genetic
variation in the genus (Duke, 1990).
Since the beginning of the molecular era,
a number of large-scale analyses of DNA
sequences from the chloroplast (cp) genome
(Wagstaff & Olmstead, 1997; Oxelman &
al., 1999; Bremer & al., 2002) and from both
the cp and nuclear genomes (Schwarzbach &
McDade, 2002) have been presented. These

Table 1. Classification of Avicennia.
Within asterids:
Verbenaceae (e.g. Schauer, 1847; Bentham, 1876; Briquet, 1895; Melchior, 1964)
Avicenniaceae (e.g. Endlicher, 1838; van Tieghem, 1898; Erdtman, 1945; Moldenke, 1960)
Outside asterids:
Dipterocarpaceae (Croizat in letters to Moldenke, cited by Moldenke, 1960)
Avicenniales near Santalales (Van Tieghem, 1898)
Celastraceae (Dahlgren, 1975)
studies all imply that Avicennia belong to the
asterids, among the Lamiales. Schwarzbach
& McDade (2002) further suggested that the
nearest relatives are found in Acanthaceae,
which is rather surprising since a placement
in this family has not been suggested in any
earlier attempts to classify the genus. They
found moderate but consistent support for a
sister relationship to the subfamily
Thunbergioideae, and this was also observed
in a recent molecular study by Borg & al. (in
press).
Acanthaceae comprise three subfamilies
(sensu Scotland & Vollesen, 2000): the
Acanthoideae include the vast majority of
genera and species and constitute a well
defined group of plants, representing
Acanthaceae sensu stricto; the Nelsoni-
oideae show some aberrant characters and
have, by some authors, been classified also
outside the Acanthaceae in the past; the
same is true for the Thunbergioideae, a
morphologically and molecularly well
supported clade, that differs in several
aspects from both the Acanthoideae and
Nelsonioideae. Despite molecular support
for a position of Avicennia among
Acanthaceae, Schwarzbach & McDdade
(2002) were unable to find morphological
support for the new findings.
Studies of floral structure and
development have helped to elucidate many
controversial interpretations of floral
structures (e.g. Endress, 2003; von Balthazar
et al., 2004; Schönenberger & Grenhagen,
2005), and have also proved valuable for
25
understanding evolutionary relationships
among Thunbergioideae (Schönenberger &
Endress, 1998). To date, no comprehensive
study of floral morphology in Avicennia has
been presented. In general descriptive and
systematic publications (e.g. Schauer, 1847;
Briquet, 1895; Cronquist, 1981; Thorne,
1992) floral morphology has not been
described in detail. Other authors studied
particular organs (e.g. wood anatomy:
Zamski, 1979 and Carlquist, 1992; pollen:
Mukherjee & Chanda, 1973 and Saxena,
1981; gynoecium: Junell, 1934; endosperm:
Padmanabhan, 1964), but usually only in
mature flowers and not from a comparative
developmental point of view. Indeed, the
need to study Avicennia in deeper detail in
order to learn more about the taxonomic
status and relationships of the genus has
been pointed out several times
(Padmanabhan, 1960; Schwarzbach &
McDade, 2002).
The aims of this study are (i) to produce
a detailed description of floral development
and structure of Avicennia and (ii) to identify
structural characters that might support a
close relationship of Avicennia with
Acanthaceae and in particular also with the
proposed sister clade Thunbergioideae.
MATERIALS AND METHODS
We studied three out of eight species of
Avicennia (sensu Tomlinson, 1995)
representing the biogeographic distribution

of the genus: A. germinans occurs along the
coasts of the Americas and the Atlantic coast
of Africa, A. bicolor occur along the pacific
coast of Central America, and A. alba occur
along the coasts of south-eastern Asia and
islands of the south-western Pacific.
The following taxa were used in this
study:
Avicennia germinans (L.) Stearn; USA;
Schönenberger 725 (voucher in ethanol) (S)
A. bicolor Standl.; Costa Rica; Borg 10 (S)
A. alba Blume; Malaysia; Rutschmann 71 (Z)
Living material fixed in FAA (formalin-
acetic acid-alcohol) and subsequently stored
in 70% ethanol was used for both microtome
sections and scanning electron microscopy
(SEM) investigations. For serial sectioning,
the material was dehydrated in graduated
series of ethanol, embedded in Kulzer’s
Technovit 7100 polymerizing resin
(Heraeus-Kulzer, Wehrheim, Germany)
following Igersheim & Cichocki (1996), cut
using a Microtom HM rotary microtome 355
(Microm, Walldorf, Germany) at 6 µm, and
stained with Ruthenium red and Toluidine
blue. For SEM studies, the specimens were
dehydrated in an ethanol series and acetone,
critical point dried, and sputter coated with
gold.
RESULTS
A detailed description of early floral
development and mature flowers is given for
A. germinans, of which we had abundant
material in various developmental stages.
For A. bicolor and A. alba only mature
flowers were studied and mainly features
deviating from those of A. germinans are
described.
Avicennia germinans
The inflorescence is apparently a thyrse
(Fig. 1M). Flowers are bisexual, basically
26
pentamerous (seemingly tetramerous in
corolla and androecium, dimerous in
gynoecium), zygomorphic, hypogynous. The
corolla is cream-white and ca 15 mm wide at
anthesis. Flowers are sessile, each is
subtended by two rather thick, keeled,
triangular bracteoles and a keeled, triangular
bract partially covering the bases of the
bracteoles.
Early floral development
Floral development begins with an
approximately triangular floral apex (Fig.
1A). The first floral organ to arise is the
dorsal (upper) sepal, which is followed by
the two ventral ones and finally by the two
lateral ones (Fig. 1A-B). The dorsal sepal is
conspicuously larger during early floral
development and later partially covers the
other four sepals (Fig 1C). As the young
flower grows, the calyx lobes elongate and
enclose the androecium and gynoecium and
will overlap in a quincuncial aestivation (see
below). After all five sepals have formed,
five petal primordia appear simultaneously
(Fig. 1D). Between the petals, five stamen
primordia are initiated simultaneously (Fig.
1D), of which the dorsal degenerates at an
early stage (Fig. 1E) and only four stamens
continue development (Fig. 1F). The two
dorsal petals eventually become united via
interprimordial growth at an early stage of
floral development (Fig.1E-F). The last
organs to appear on the floral apex are the
two carpels, which arise as a congenitally
united mound in the centre of the flower (Fig
1F).
Morphology and anatomy of mature flowers
The abaxial surface of bract and
bracteoles are covered by trichomes (Fig.
3A) and scattered glandular hairs. The
glandular hairs are partly sunk into the
epidermis and consist of a short one-celled
stalk and several secretory cells clustered in
a head-like structure (Fig. 3B). On the
adaxial side of the bract glandular hairs are
 27

Fig. 1. Floral development and morphology in Avicennia. A-L: Avicennia germinans. A-C, calyx development (bract
and bracteoles removed); D-F, corolla and androecium development (calyx removed); G, stamen; H, glandular hairs
on filament base; I, gynoecium; J-K, ovule structure, micropyle indicated by arrow head; L, flower. M: A. bicolor,
inflorescence. A-F scale bars = 200µm, G-K scale bars = 100 µm. Abbreviations: C = carpel; P = petal lobe; S =
sepal lobe; St = stamen.

Fig. 2. Line drawing of serial transverse sections of floral bud of Avicennia germinans. A-C, free parts of petals; D-
F, corolla tube; G-J, stamen-corolla tube; K-L, floral base. Scale bar = 1 mm.
sparse. Both the bract and bracteoles have a
thick median as well as several smaller
lateral vascular bundles (Fig. 2D-G). The
calyx consists of five broad-based, largely
distinct (united only at their very base, Fig.
2I-L), ovate sepals that are quincuncially
arranged in bud (Fig. 2E-I). Sepals are
covered by trichomes as well as glandular
hairs abaxially but are almost completely
glabrous adaxially with just a very few
scattered glandular hairs. Sepal
vascularization consists of a relatively large
median bundle and several smaller lateral
bundles (Fig. 2D-J). The lateral bundles of
two neighbouring sepals form synlaterals in
the short synsepalous zone close to the floral
base (Fig. 2K), where the five median and
the five synlateral calyx traces enter the
central vascular cylinder (Fig. 2L). The
corolla is sympetalous in the lower half, with
28
the four lobes having a left contort
aestivation in bud (Fig. 2A). The dorsal lobe,
that consists of two united petals, is weakly
emarginate and broader than the ventral and
two lateral ones. Lateral and ventral corolla
lobes contain one major median and many
smaller lateral vascular bundles (Fig. 2B-C).
In the dorsal lobe the vascular system
consists of two major and a series of smaller
bundles, reminiscent of its bipetalous nature
(Fig. 2C). In the corolla tube, lateral bundles
merge with each other and further down in
the tube they form five distinct groups close
to the five median bundles (Fig. 2G-H). At
the very base of the tube, each of the five
groups of the bundles forms a single median
strand (Fig. 2J-K) that joins the central
vascular cylinder in the floral base (Fig. 2L).
on the abaxial surface (Fig. 3C). Numerous
glandular hairs are present on both surfaces
 29

Fig. 3. Transverse sections of floral organs of Avicennia. A-G: Avicennia germinans. A, abaxial side of bracteole
with trichomes; B, glandular hairs on bracteole; C, abaxial side of corolla with trichomes and glandular hairs; D,
immature anthers with pollen sac placentoids (arrow heads); E, ovary with short uniseriate trichomes; F, detail of
uniseriate trichomes on gynoecium; G, unequal stylar lobes, adaxial stigmatic surface is weakly papillate (arrow
heads). H-I: A. bicolor. H, lower part of gynoecium with trichomes and glandular hairs; I, detail of trichomes and
glandular hairs of gynoecium. J-N: A. alba. J, almost equal, slightly overlapping corolla lobes and four equal
stamens; K, corolla lobes having left contort aestivation; L, upper part of gynoecium with glandular hairs and
trichomes; M, middle part of gynoecium with glandular hairs; N, lower part of gynoecium glabrous. A-I, L-N scale
bars = 100 µm; J-K scale bars = 1 mm.

more common abaxially. The four stamens
are exserted from the corolla tube and
slightly unequal in length with the two
ventral ones somewhat longer (Fig. 2B-C).
The filament bases are weakly thickened at
the level of their attachment to the corolla
tube (Fig. 2G) and have glandular hairs (Fig.
1H). Anthers are sagittate, basifixed and
have a slightly rounded outline (Fig. 1G).
The thecae open introrsely by longitudinal
slits. Pollen sac placentoids are visible in
immature anthers (Fig. 3D). An endothecium
is differentiated and extends into the
connective tissue (Fig. 3D). Each stamen is
served by a single vascular bundle passing
through the stamen-corolla tube and directly
entering the central vascular cylinder in the
floral base (Fig. 2C-L). No stamen bundle is
present on the dorsal side of the flower
(fifth, degenerate stamen). The gynoecium is
surrounded by a shallow, not-vascularized
nectary disk (Fig. 2J). The syncarpellate
gynoecium is flask shaped and covered by
short uniseriate trichomes and scattered
glandular hairs (Fig. 1I, Fig. 3E-F). Few
glandular hairs are also present within the
ovary (not shown). The narrow stigmatic
lobes are glabrous, and the ventral lobe is
slightly shorter than the dorsal one. The
stigma that covers the adaxial surfaces of the
two lobes is weakly papillate (Fig. 3G). Each
carpel produces two collateral ovules that are
attached to the top of a massive central
column rising from the base of the ovary
(Figs. 1J-K, 2F). The unitegmic ovules are
pendulous and orthotropous with the
micropyle directed to the outside of the basal
part of the ovary (Fig. 1J-K). The ovary is
basically unilocular, i.e. the central column
is free from the carpel walls over most of its
length (Fig. 2G-H). A short septum is
present only at the very base of the ovary
(Fig. 2I). Each carpel contains a dorsal and
several smaller lateral vascular bundles (Fig.
2D-J), of which only the two dorsal ones are
present in the uppermost part of the style
(Fig. 2C). The four ovules are served by a
30
massive central vascular strand that passes
through the central column of the ovary and,
in the floral base, is directly attached to the
central vascular cylinder (Fig. 2F-K).
Avicennia bicolor
Morphology and anatomy
General floral organization and structure
is identical to that of A. germinans. The
corolla is white with a yellow throat and
smaller than A. germinans, ca 6 mm wide at
anthesis. The flowers have shorter stamens
and style than A. bicolor. The two bracteoles
are obtuse, the bract is short and rounded. As
in A. germinans, the abaxial surfaces of bract
and bracteoles are covered by trichomes and
scattered glandular hairs. The ventral and
two lateral corolla lobes are obtuse. The
adaxial lobe is broader than the others and is
itself shortly two-lobed, as in A. germinans.
Calyx and corolla are covered by trichomes
and bear glandular hairs on the abaxial
surface. The four stamens are inserted at the
same level, but are unequal: The ventral
stamens are curved upwards to the dorsal
side of the flower and all four anthers are
positioned beneath the adaxial lobe. At
anthesis, they reach just above the level at
which the petal lobes spread. The gynoecium
is covered by long trichomes and abundant
glandular hairs (Fig. 3H-I). A few glandular
hairs are present inside the ovary, and the
style has two unequal lobes, as in A.
germinans.
Avicennia alba
Morphology and anatomy
Unlike A. germinans and A. bicolor, the
flowers of A. alba are actinomorphic at
anthesis (Fig. 3J) with apparently equal petal
lobes, and have a yellow corolla which is ca
3 mm in diameter at anthesis. The bracteoles
are triangular, the bract is large, thick and
triangular. As in A. germinans and A.
bicolor, the bract has trichomes and

scattered glandular hairs abaxially, but
unlike the two other species the two
bracteoles are glabrous. The calyx lobes are
ovate. The corolla lobes appear almost
valvate in bud, only slightly overlapping in a
contort arrangement (Fig. 3J-K). The corolla
is covered by trichomes and glandular hairs
on outside as in the other examined species,
but the sepals are glabrous. The four stamens
alternate with the corolla lobes and are
inserted at the same level on the corolla tube.
They are not exserted at anthesis. The
filament bases bear glandular hairs as in A.
germinans (Fig. 1H). The upper part of the
gynoecium (except the style) is covered by
trichomes and glandular hairs (Fig. 3L),
while the middle is covered only by
abundant glandular hairs (Fig. 3M) and the
lower part is completely glabrous (Fig. 3N).
No glandular hairs are present inside the
ovary. The style is very short, glabrous and
positioned below the anthers. Unlike A.
germinans and A. bicolor, A. alba has equal
stylar lobes.
DISCUSSION
As in the majority of the Acanthaceae,
belonging to the subfamily Acanthoideae,
the inflorescences of Avicennia are
apparently thyrses (Fig. 1M) with the
individual cymes often reduced to a single
flower (Schwarzbach & McDade, 2002).
The flowers of Acanthoideae and
Nelsonioideae are usually subtended by a
bract and two bracteoles (prophylls), and this
applies also to Avicennia, while in
Thunbergioideae the bract is absent
(Schwarzbach & McDade, 2002).
General floral structure
As early developmental stages reveal, the
flowers of Avicennia have a pentamerous
floral ground plan with a dimerous
gynoecium. This ground plan is not only
found in all other Acanthaceae, but also in
31
the majority of the other families of the
Lamiales. Likewise are sympetaly and
zygomorphy, as present in the flowers of
Avicennia (Fig. 1L), characteristic features
of the Lamiales. In correlation with
zygomorphy, a strong reduction or even a
complete loss of the median (dorsal) stamen
is also widespread in Lamiales (Endress,
1994). Another characteristic feature of the
Lamiales that is also present in Avicennia,
but occurs in only few other angiosperms,
are pollen sac placentoids in the anthers
(Hartl, 1963), a connective protrusion of
parenchymatic tissue into each pollen sac
(Fig. 3D).
Calyx
The pentamerous calyx of Avicennia is
functional during floral development, i.e. it
encloses and protects the other floral organs
during development. It shows a quincuncial
aestivation pattern. This is also found in
many Acanthoideae and Nelsonioideae
(Reddy & al., 1980), whereas in
Thunbergioideae the calyx is strongly
reduced, usually represented only by a small
rim or by a number of small lobes.
Protection of developing floral organs of
thunbergioid flowers is provided by the
enlarged bracteoles (Schönenberger and
Endress, 1998). Calyx aestivation is poorly
documented for Nelsonioideae, but is
descending cochlear in the nelsonioid
species Elytraria crenata and reduced to
four sepals by fusion in Nelsonia campestris
(Reddy & al., 1980). Both in Avicennia
(Sanders, 1997) and in Thunbergioideae
(personal observation) the calyx is persistent
after anthesis, but this is also common in
other Acanthaceae (e.g. Wasshausen &
Wood, 2004).
Corolla
The corolla in all three species has
apparently four lobes, but this is only a
superficial reduction from a pentamerous
organization, as seen in early developmental

stages (Fig. 1D-F). The dorsal lobe in A.
germinans and A. bicolor is slightly two-
lobed and broader at the base also in the
mature flower (fig. 1L), and has more
vascular bundles than the ventral and lateral
lobes (fig. 2C-D). Tomlinson (1995) reports
that five-lobed corollas are occasionally
present in some populations of A. marina. A
reduction from five to four petals has
evolved in parallel in several other
representatives of families of the Lamiales
(Endress, 1994). Corolla aestivation is left
contort in Avicennia (Fig. 2A), as found in
many Thunbergioideae and also in some
Acanthoideae, while in Nelsonioideae it is
descending cochlear (Scotland & Vollesen,
2000).
Androecium
Five stamen primordia are visible in
Avicennia but only four develop (Fig. 1D-F),
as has been shown to be the case also in
Thunbergioideae (Schönenberger & Endress,
1998; Schönenberger 1999). Most
Acanthoideae also have four functional
stamens, while in Nelsonioideae, which also
have a basically pentamerous androecium,
the number of stamens is reduced to only
two (Reddy et al., 1980). A study of
vasculature in Acanthaceae (Rao, 1953)
show that in genera with four stamens, five
vascular traces for the androecium are
present, of which one is smaller and
disappears at the level of insertion of the
stamens. The filament bases in Avicennia are
swollen, and bear glandular hairs (Fig 1G-
H). Both these characteristics are also found
in Thunbergioideae (Schönenberger, 1999).
In immature anthers of Avicennia pollen sac
placentoids are visible (Fig. 3D), a feature
present also in Thunbergioideae (Schönen-
berger & Endress, 1998), but as discussed
above they occur as well in other
Acanthaceae and other Lamiales. The shape
and structure of stamens differs from that of
Thunbergioideae. In Avicennia, the endo-
thecium is well developed and the thecae
32
open by longitudinal dehiscence (Fig. 1G),
as in most Acanthaceae, while
Thunbergioideae are exceptional in lacking
differentiated endothecium cells and have
thecae which open by long or short slits or
even pores (Schönenberger & Endress
1998). Furthermore, connective tips and
anther appendages such as bristles or awn-
like structures are common in Thun-
bergioideae while in Avicennia anthers are
blunt.
Pollen grains in Avicennia are spheroidal
and tricolporate, with longitudinally
elongated ora and reticulate surface
ornamentation with broad muri and small
lumina (Mukherjee & Chanda, 1973;
Chadwell & al., 1992). Pollen morphology
varies enormously in Acanthaceae, regarding
e.g. number of apertures and surface
ornamentation, and this makes it problematic
to use pollen morphological characters for
analysing relationships among larger groups
in the family (Scotland & Vollesen, 2000).
Nor has it been possible to relate pollen
morphology in Avicennia to any other group
among Lamiales, although Mukherjee
(1973b) suggested the genus to be closely
related to Phryma and Myoporaceae. This
was later rejected by Chadwell & al. (1992).
Gynoecium
The gynoecium in Avicennia is
bicarpellate and produces four ovules (two
per carpel), as is common in Lamiales. They
are pendent from the top of a free central
column (Fig. 1J-K). Such a free central
column is not present in any other
Acanthaceae, but a similar type of ovary
structure with a free central placenta exists
in two families of Lamiales (Scrophulari-
aceae and Lentibulariaceae; Hartl, 1956),
which, however, are only distantly related to
Avicennia. Ovules are arranged collaterally,
i.e., side by side (Fig. 2G-I), as in
Thunbergioideae (Schönenberger & Endress,
1998) and other Acanthaceae, as well as in
Verbenaceae, to which several earlier

authors have linked Avicennia (e.g.
Bentham, 1876; Briquet, 1895). Verbena-
ceae and Lamiaceae both have ovaries with
four ovules, divided into four locules
because of the development of a false
septum (Wagstaff and Olmstead, 1997).
Sanders (1997) argues that the basal partition
of the ovary and the apex of the central
column in Avicennia are directly comparable
to the complete partitions dividing the ovary
in Verbenaceae and Lamiaceae, although in
Avicennia the ovules are apical, pendulous,
and more or less orthotropous. The
descriptions in the literature of ovule
orientation in Verbenaceae are not
consistent. Thunbergioideae and Acantho-
ideae have anatropous ovules, while ovules
in Nelsonioideae are many and have been
described as being amphitropous (Johri &
Singh, 1959). However, this character is
very variable among Lamiales and the
phylogenetic utility is uncertain (Schwarz-
bach & McDade, 2000). Furthermore,
Padmanabhan (1960) claim that the
condition in A. officinalis should be
interpreted as an “arrested stage in the
development of an anatropous ovule” only
superficially ressembling an orthotropous
condition with micropyles enclosed in the
partial basal locules. Placentation is
essentially axile in Avicennia, as in
Acanthaceae, but also Verbenaceae and
Pedaliaceae (Schwarzbach & McDade,
2002).
The style is two-lobed, as in most
Acanthaceae, and the lobes are unequal in A.
germinans and A. bicolour (Fig. 3G).
Unequal lobes are a characteristic feature for
Thunbergioideae (Schönenberger & Endress,
1998) and also occur in some Acanthoideae.
Two-lobed styles are present also in other
families of Lamiales, and the lobes may also
be unequal.
The embryology of various
representatives of Acanthaceae as well as of
Avicennia has been described by various
authors. In the majority of Acanthaceae,
33
including Nelsonioideae but not Thun-
bergioideae, the first two divisions of the
endosperm are transverse and result in a
primary 3-celled endosperm, of which the
middle cell develops into the “endosperm
proper” while the other two cells develop
into micropylar and chalazal haustoria
(Mohan Ram & Wadhi, 1965). In
Thunbergioideae the chalazal haustorium is
absent; instead the chalazal chamber forms
the endosperm with many secondary
haustoria (Maheshwari & Kapil, 1966). The
endosperm initiation in Avicennia, described
by Padmanabhan (1964), appears to develop
as in Acanthoideae and Nelsonioideae. After
formation, the endosperm in Avicennia is
devoid of reserve food material and
functions only as a conductive tissue
transporting nutrients to the suspensor
(Maheshwari & Kapil, 1966), and the
endosperm has therefore been described as
being absent (e.g. Sanders, 1997). The
endosperm in Acanthoideae and Thun-
bergioideae is not persistent, and seeds lack
endosperm, while Nelsonioideae have oily
endosperm (Schwarzbach & McDade, 2002).
An endosperm development similar to that
found in Acanthoideae, Nelsonioideae and
Avicennia is found in some Lamiaceae, but
is likely a parallelism (Stevens, 2001).
However, endosperm is also lacking in most
Verbenaceae, Lamiaceae and some
Pedaliaceae (Schwarzbach & McDade,
2002).
The fruit in Avicennia is usually an
asymmetric capsule, ovoid and elongated
towards the apex (persistent stylar beak)
with a dry or to some extent fleshy exocarp.
The capsules are usually one-seeded as the
other three ovules abort, and dehisce by two
thickened valves (Moldenke, 1960). The
fruit characters are partially reminiscent of
the two fruit types found in
Thunbergioideae: in Thunbergia and
Pseudocalyx fruits are four-seeded,
dehiscent capsules with a prominent stylar
beak; in Mendoncia, fruits are indehiscent

drupes with one- or rarely two seeds
(Benoist, 1944; Schönenberger & Endress,
1998).
Indumentum
Glandular hairs seem to occur on leaves
in all species of Acanthaceae (Ahmad,
1978). Subsessile (short stalked) glandular
hairs with panduriform heads are recorded
for Thunbergioideae and Nelsonioideae
while Acanthoideae have globular heads
(Ahmad, 1978). The flowers of Avicennia
apparently have the same kind of glandular
hairs as are found on leaves in Acanthoideae.
At least some species of Avicennia have
scented flowers (e.g. A. alba and A.
germinans; Moldenke, 1960). Aromatic
gland-headed hairs occur also in
Verbenaceae and Lamiaceae and have
probably evolved more than once within
Lamiales (Wagstaff and Olmstead, 1997).
Vegetative characters
Stem nodes of Avicennia are articulated
as in most Acanthaceae, including
Thunbergioideae but excepting Nelsoni-
oideae and some Acanthoideae. Schwarz-
bach & McDade (2002) suggested this could
be a synapomorphy for Acanthoideae and
Thunbergioideae plus Avicennia.
CONCLUSIONS
The black mangrove genus Avicennia
shares many general similarities with other
members from various groups of the
Lamiales, and also presents several
apparently autapomorphic characters, such
as orthotropous ovules pendent from a free
central column. Consequently, previous
morphological studies have failed to uncover
undisputable synapomorphic characters
linking this group to a specific family. This
study documents a number of similarities
between Avicennia and the acanthaceous
subfamily Thunbergioideae, including a
34
persistent calyx, left contort corolla
aestivation, presence of five stamen
primordia of which one degenerates,
thickened filament bases, glandular hairs on
filament bases, presence of pollen sac
placentoids, collateral ovule arrangement,
two ovules per carpel, and unequal stigma
lobes. Other similarities include articulated
stems and aspects of fruit morphology.
However, several of these characters are also
shared by other clades of the Acanthaceae,
and some are also commonly found in
various other groups of the Lamiales. Based
on morphological data, a placement among
the Acanthaceae is supported, but at present,
there is not enough evidence to claim a
closer relationship of Avicennia to
Thunbergioideae than to any other
acanthaceous lineage.
ACKNOWLEDGEMENTS
We thank Luis Diego Gomez, Eugenio
González and Francisco Campos from the
Organization for Tropical Studies (OTS) for
help with collection permits in Costa Rica.
For help with the microtome, we thank
Linda Lundmark. For providing valuable
material, we are grateful to Frank
Rutschmann.
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