Annals of Botany 103: 1049– 1064, 2009
doi:10.1093/aob/mcp048, available online at www.aob.oxfordjournals.org
Woodiness within the Spermacoceae – Knoxieae alliance (Rubiaceae): retention
of the basal woody condition in Rubiaceae or recent innovation?
Frederic Lens1,*, Inge Groeninckx1, Erik Smets1,2 and Steven Dessein3
1
Laboratory of Plant Systematics, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31 Box 2437,
BE-3001 Leuven, Belgium, 2Nationaal Herbarium Nederland – Leiden University Branch, P.O. Box 9514, NL-2300 RA Leiden,
The Netherlands and 3National Botanic Garden of Belgium, Domein van Bouchout, Nieuwelaan 38, BE-1860 Meise, Belgium
Received: 12 November 2008 Returned for revision: 11 December 2008 Accepted: 16 January 2009 Published electronically: 11 March 2009
† Background and Aims The tribe Spermacoceae is essentially a herbaceous Rubiaceae lineage, except for some
species that can be described as ‘woody’ herbs, small shrubs to treelets, or lianas. Its sister tribe Knoxieae contains a large number of herbaceous taxa, but the number of woody taxa is higher compared to Spermacoceae. The
occurrence of herbaceous and woody species within the same group raises the question whether the woody taxa
are derived from herbaceous taxa (i.e. secondary woodiness), or whether woodiness represents the ancestral state
(i.e. primary woodiness). Microscopic observations of wood anatomy are combined with an independent molecular phylogeny to answer this question.
† Methods Observations of wood anatomy of 21 woody Spermacoceae and eight woody Knoxieae species, most
of them included in a multi-gene molecular phylogeny, are carried out using light microscopy.
† Key Results Observations of wood anatomy in Spermacoceae support the molecular hypothesis that all the
woody species examined are secondary derived. Well-known wood anatomical characters that demonstrate this
shift from the herbaceous to the woody habit are the typically flat or decreasing length vs. age curves for
vessel elements, the abundance of square and upright ray cells, or even the (near-) absence of rays. These socalled paedomorphic wood features are also present in the Knoxieae genera Otiophora, Otomeria, Pentas,
Pentanisia and Phyllopentas. However, the wood structure of the other Knoxieae genera observed
(Carphalea, Dirichletia and Triainolepis) is typical of primarily woody taxa.
† Conclusions In Spermacoceae, secondary woodiness has evolved numerous times in strikingly different habitats. In Knoxieae, there is a general trend from primary woodiness towards herbaceousness and back to (secondary) woodiness.
Key words: Knoxieae, LM, primary woodiness, Rubiaceae, Rubioideae, secondary woodiness, Spermacoceae,
wood anatomy.
IN T RO DU C T IO N
In its currently accepted circumscription, the tribe
Spermacoceae is essentially a herbaceous lineage of the
family Rubiaceae, representing about 61 genera and 1235
species (Kårehed et al., 2008; Groeninckx et al., 2009a). It
includes the former tribes Spermacoceae sensu stricto and
Manettieae, and the Hedyotis – Oldenlandia group of the
former tribe Hedyotideae. Representatives are usually
annuals or short-lived perennials found in grasslands or open
forests throughout the (sub)tropics. A number of species are
adapted to more extreme habitats, such as sand dunes,
Kalahari sand plateaus (Fig. 1A), shores (Fig. 1B, C),
montane scrublands or rocky areas (Table 1). Many species
are entirely herbaceous, but several (short-lived) perennial
species – especially those adapted to extreme habitats –
produce a limited-to-considerable amount of wood (‘woody’
herbs to small shrubs, or occasionally treelets, Fig. 1D – F)
while others develop secondary growth mainly in their underground organs (geoxylic herbs, Fig. 1A– C; Dessein et al.,
2002, 2003). The occurrence of herbaceous and woody
species within the same group raises the question whether
* For correspondence. E-mail frederic.lens@bio.kuleuven.be
the herbaceous taxa are derived from woody lineages, as is
the case in the early-diverging lineages of the subfamily
Rubioideae (e.g. in Coccocypselum and Cruckshanksia of
the tribe Coussareeae sensu lato), or if herbaceousness represents the plesiomorphic state. From an evolutionary point
of view, the second hypothesis refers to a secondary derived
origin of the wood, meaning that these woody species are
derived from herbaceous ancestors, which in turn have
evolved from ( primarily) woody species.
With respect to higher-level phylogenetic relationships of
Spermacoceae, the tribe is considered as a derived taxon
within Rubioideae and there is strong evidence for a sister
relationship with Knoxieae (Bremer and Manen, 2000;
Dessein, 2003; Robbrecht and Manen, 2006). The Knoxieae
as currently accepted are a much smaller tribe than
Spermacoceae: they include only 17 genera and 128 species,
and unite Knoxieae s.s., Triainolepideae and members of the
Pentas group of the former tribe Hedyotideae (Dessein, 2003;
Kårehed and Bremer, 2007). Knoxieae generally differ from
Spermacoceae in having predominantly 5-merous flowers
instead of 4-merous flowers, but exceptions occur in both
tribes. As in Spermacoceae, herbaceous and woody species
are present in Knoxieae, although the percentage of woody
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Lens et al. — Woodiness in Spermacoceae – Knoxieae
A
B
C
D
E
F
F I G . 1. Examples of the variation in habitats and growth forms of woody Spermacoceae. (A, B) habitats plus growth form; (C– F) growth form. (A) Overview of
Kalahari sand plateau (Mwinilunga, Zambia) where Spermacoce manikensis grows (geoxylic herb; inset, detail of habit). (B) Shore of Lake Bangweulu (Zambia)
with S. bangweolensis (inset, detail of habit). (C) Gomphocalyx herniarioides, geoxylic herb (south-west Madagascar). (D) Lathraeocarpa acicularis, shrub
(south-west Madagascar). (E) ‘Oldenlandia’ ambovombensis, shrub (south Madagascar). (F) ‘Oldenlandia’ humbertii, shrub (south-west Madagascar).
representatives is clearly higher. For instance, the genera
Carphalea, Dirichletia and Triainolepis are entirely woody
and also usually taller than most woody Spermacoceae. In
most remaining Knoxieae genera, some of the species produce
a limited amount of wood and can be described as small (sub)shrubs, woody herbs or geoxylic herbs (cf. most woody
Spermacoceae; Verdcourt, 1976; Table 1).
The intratribal relationships of Spermacoceae have been the
subject of controversy. Continuous changes in the delimitation
of genera have resulted in a very complex taxonomic history of
the tribe. Recent molecular studies (Kårehed et al., 2008;
Groeninckx et al., 2009a) have shed new light on the phylogeny
of Spermacoceae and relationships between genera are starting
to crystallize. The genera of the Spermacoceae s.s. form a
monophyletic clade deeply nested among taxa of the Hedyotis–
Oldenlandia group, and are closely related to Bouvardia and
Manettia. Oldenlandia is polyphyletic and should be restricted
to include only close relatives of its type species O. corymbosa.
Hedyotis is possibly to be restricted to Asia. Many smallto-medium-sized genera (e.g. Arcytophyllum, Conostomium,
Hedythyrsus, Houstonia, Stenotis, etc), often reduced to the synonymy of either Hedyotis or Oldenlandia, should be recognized at
generic level. With respect to the Knoxieae, generic rearrangements have been proposed by a molecular study of Kårehed and
Bremer (2007). For instance, Pentas was shown to be polyphyletic
and three new genera were recognized to accommodate its
species, Placopoda and African members of the genus Carphalea
should be placed into Dirichletia, and the generic circumscription
TA B L E 1. Habit and habitat details of the woody species studied
Species
Emmeorhiza umbellata
Gomphocalyx herniarioides
Hedyotis flavescens
Hedyotis fruticosa
Hedyotis lessertiana
‘Hedyotis’ trichoglossa
Kadua cordata
Lathraeocarpa acicularis
Mitracarpus frigidus
Nesohedyotis arborea
‘Oldenlandia’
ambovombensis
‘Oldenlandia’ humbertii
Spermacoce bangweolensis
Spermacoce manikensis
Spermacoce macrocephala
Spermacoce occidentalis
Spermacoce verticillata
Knoxieae
Carphalea kirondron
Dirichletia virgata
Otiophora rupicola
Otomeria micrantha
Pentanisia schweinfurthii
Pentas zanzibarica
Phyllopentas schimperiana
Triainolepis polyneura
Subshrub
Shrub
Subshrub or shrub
Straggling, scrambling or climbing, often perennial herb
Climbing, perennial herb
Prostrate or decumbent herb with well-developed, often
woody taproot
Shrub
Shrub or small tree to 4 m
Shrub or small tree to 4 m
Woody herb of suffrutex with slightly woody stems and
taproot
Subshrub or shrub
Subshrub with woody stems and taproot
Subshrub or shrub
Tree up to 7 m
Shrub
Habitat and distribution
Volcanic mountains at high altitude; Costa Rica to Panama
Grassland above timberline; western South America
Grassland above timberline; Colombia to Peru
Large ecological amplitude, including evergreen forest, riverine vegetation, bush land, rocky places;
widespread in the tropics
Tropical forest; South America
Sandy soils of thorn forest, dunes and beaches; Madagascar
Upper montane zone on open waterlogged sites; Sri Lanka
Open vegetation, rocky areas on mountain tops; southern India, Sri Lanka, Myanmar
Upper montane scrubland; Sri Lanka
Eastern humid forest; Madagascar
Mesic to wet forests; Hawaiian islands
Sandy soils in dunes close to the sea; Madagascar
Scrubland, grassland, also in disturbed areas; tropical America
Tree fern thicket; St. Helena island
Dry spiny forest on limestone plateau; south-western Madagascar
Subshrub
Prostrate or suberect subshrub with woody taproot
Geoxylic herb with woody taproot and plant base
Subshrub
Annual or more often perennial spreading or erect woody
herb
Woody herb or (sub)shrub
Dry spiny forest on limestone plateau; south-western Madagascar
Sandy soil bordering Lake Bangweulu; Zambia
High plateaus on Kalahari sand; Zambia and D.R. Congo
Savanna areas on river banks; Colombia and Venezuela
Open woodland on sandy soil or dune vegetation close to sea; Australia
Shrub or tree up to 10 m
Shrub
Subshrub
Herb, often with woody base
Geoxylic herb with woody taproot and plant base
Woody herb or shrub with a woody rootstock
Woody herb or shrub
Shrub
Mostly at edges of dry, deciduous forest on laterite or calcareous sand; Madagascar
Forest and scrubland; Socotra, Republic of Yemen
Rocky grassland at high altitude; Burundi
Savanna, clearings in forest, along roadsides; Nigeria to west-central tropical Africa
Submontane and lower grassland, also in clearings of woodland; tropical Africa
Forest edge, scrubland and grassland; Uganda to Mozambique
Forest edge and scrubland, at high altitudes; tropical Africa
Deciduous and semi-deciduous forest; Madagascar
Savanna, scrubland and disturbed areas; tropical and subtropical America, introduced elsewhere
Lens et al. — Woodiness in Spermacoceae – Knoxieae
Spermacoceae
Arcytophyllum lavarum
Arcytophyllum setosum
Arcytophyllum thymifolium
Diodella sarmentosa
Habit
1051
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Lens et al. — Woodiness in Spermacoceae – Knoxieae
of Pentanisia and Triainolepis must be enlarged. The current molecular framework of Spermacoceae (sensu Kårehed et al., 2008;
Groeninckx et al., 2009a) and Knoxieae (Kårehed and Bremer,
2007) forms a good starting point to assess the origin of woodiness
within both tribes.
In order to evaluate secondary woodiness of a particular
species, independent methods should be applied. If a molecular phylogeny is present, the first and most obvious way is to
trace evolutionary shifts from herbaceous to woody taxa (e.g.
Böhle et al., 1996; Thiv et al., 1999; Lee et al., 2005).
Another option might be to make woody mutants from herbaceous wild-types (Groover, 2005; Melzer et al., 2008). If molecular data are insufficient or unavailable, evidence in the
microscopic wood structure – the so-called paedomorphic features – can be used to identify secondary derived shrubs and
trees (Carlquist, 1962). Paedomorphic features typically
remain in a juvenile ontogenetic state, which can be demonstrated in the wood structure by ( juvenile) characters of the
primary xylem that are transferred into the (mature) secondary
xylem (¼ wood). Examples are the continuous decrease of
vessel element length from the pith towards the cambium,
the presence of elongated scalariform intervessel pits in
wood (or even wide, gaping intervessel pits resembling
helical tracheids in the primary xylem; e.g. fig. 7 in Lens
et al., 2005), and the absence of rays and/or the presence of
rays with an abundance of square-to-upright ray cells.
In at least some (derived) Rubioideae lineages with mixed
herbaceous and woody species, such as Paederieae and
Rubieae, earlier publications of wood anatomy favour the secondary woodiness option for at least some species
(Koek-Noorman, 1976; Koek-Noorman and Puff, 1983;
Carlquist, 1992). Consequently, it would be interesting to
investigate if this is also valid in other derived Rubioideae
clades, such as Spermacoceae and its sister tribe Knoxieae.
In order to do this, the wood anatomical variation of
Spermacoceae and Knoxieae should be studied using a
broader sampling than is currently available. The poor knowledge of wood anatomy of both tribes can be illustrated by two
large-scale wood anatomical review papers in Rubiaceae: the
first one covering the subfamily Rubioideae (Jansen et al.,
2001) studied only two young twigs of Triainolepis
(Knoxieae), while the subsequent wood anatomical survey
concerning the entire family (Jansen et al., 2002) dealt with
mainly juvenile material of only six Spermacoceae species
and six Knoxieae species.
The objectives of this study were to present a detailed overview of the wood anatomy of woody representatives of the
tribe Spermacoceae and Knoxieae, and to compare this overview with a multi-gene molecular phylogeny in order to
assess the origin of woodiness in the two tribes. In addition,
a comparison is made of the wood structure between
Spermacoceae and its sister tribe Knoxieae.
M AT E R IA L S A ND M E T HO DS
Plant material
Wood samples from 21 Spermacoceae species and eight
Knoxieae species were collected from the xylaria in Kew
(Kw), Tervuren (Tw), Utrecht (Uw) and from the National
Botanic Garden of Belgium (BR; see Table 2 and
Appendix). The sampling covers all major Spermacoceae
and Knoxieae clades as identified in the molecular phylogenies
(Kårehed and Bremer, 2007; Kårehed et al., 2008; Groeninckx
et al., 2009a).
We tried to investigate as many mature wood samples as
possible, but due to the limited wood production in some
species, some samples need to be considered as juvenile
twigs, for example Arcytophyllum lavarum, A. thymifolium,
Emmeorhiza umbellata, Hedyotis flavescens, H. fruticosa,
H. lessertiana and ‘Oldenlandia’ humbertii
(all
Spermacoceae). The Knoxieae species Carphalea kirondron,
Otiophora rupicola and Phyllopentas schimperiana also represent juvenile wood material.
Geoxylic Spermacoceae species with woody underground
organs are observed in Gomphocalyx herniarioides and
Spermacoce manikensis; of these, G. herniarioides has
primary xylem in the centre, while the underground parts of
S. manikensis has a stem anatomy. Species with a similar
geoxylic habit in Knoxieae are Otiophora rupicola and
Pentanisia schweinfurthii. Spermacoce bangweolensis and
S. occidentalis have woody underground parts combined
with more elaborate woody parts above ground.
‘Oldenlandia’ humbertii and ‘Oldenlandia’ ambovombensis
represent two undescribed species from Madagascar: both will
be described in a new genus (I. Groeninckx, unpubl. data). The
Malagasy species ‘Hedyotis’ trichoglossa also needs to be
transferred to a new genus. Lathraeocarpa acicularis, a
small (sub)shrub also endemic to Madagascar, was previously
placed within the monogeneric tribe Lathraeocarpeae
(Bremekamp, 1957); however, a recent molecular –
morphological study by Groeninckx et al. (2009b) strongly
supports the inclusion of this Malagasy endemic genus
within the tribe Spermacoceae, sister to Phylohydrax and
Gomphocalyx.
Description of wood anatomy and microtechniques
The methodology of wood sectioning and the subsequent
steps are described in Lens et al. (2005). The wood anatomical
terminology follows IAWA list of microscopic features for hardwood identification (IAWA Committee, 1989), except for the
concept of imperforate tracheary elements with distinctly bordered pits (i.e. fibre-tracheids and tracheids). Because the distinction between these two cell types is very hard to make in
Spermacoceae due to continuous overlap in pit border size
and pit density, we prefer to distinguish between fibre-tracheids
and tracheids based on the degree of vessel grouping (Carlquist,
1984). In general, species with a low vessel grouping that grow
in mesic conditions possess non-conducting fibre-tracheids (or
libriform fibres) in the ground tissue, but when these species
occur in dry conditions the imperforate cells are hypothesized
to be functionally water-conducting tracheids that take over
the water transport from drought-induced embolized vessels
(although we acknowledge that more functional work remains
to be done to assess the difference in water-conducting capacity
between fibre-tracheids and tracheids).
For length vs. age curves, measurements for vessel elements
were made using radial sections from the pith towards the
cambium. In addition, the length of vessel elements and
Tangential vessel multiples
Vessel clusters
Occasional septate
libriform fibres
Dif axial parenchyma
Dia axial parenchyma
Banded axial parenchyma
Scanty axial parenchyma
Rays absent
Rays exclusively uniseriate
Multiseriate ray width (no. cells)
Multiseriate ray height (mm)
Ray cells mostly
or all square-to-upright
þ
þ
þ
+
2
+
2
+
+
+
+
2
+
þ
2
2
2
þ
2
+
+
+
+
2
2
2
þ
2
2
2
2
2
2
2
+
+
2
2
2
2
2
+
+
2
2
2
2 15–20–30 100– 125 –160 150 –195– 250
250 –300– 350 2
2 15–20–25 80– 100 –140 150 –290– 400
300 –370– 450 2
2 10–16–20 480– 540 –640 100 –160– 250
150 –200– 250 2
+ 25– 60–120
36–48– 66 150 –300– 550
350 –465– 550 2
2 15– 55–120
60–81– 85 200 –350– 500
500 –610– 700 2
2 15–28–40 135– 150 –180 100 –160– 250
250 –340– 400 2
2 10–23–30 90– 110 –140 300 –650– 850 750– 910–1150 2
2 25–36–50
80–95–120 250 –480– 700
600 –765– 900 2
2 15–25–30
75–45–120 300 –660– 1000 850– 1050–1300 2
2 15–21–30 100– 120 –145 300 –540– 700
500 –665– 800 2
2 20–29–50 140– 170 –230 350 –450– 700
500 –630– 800 2
+ 10–21–40 200– 255 –290 100 –220– 300
400 –470– 600 +
2 15–25–45 140– 170 –200 250 –375– 550
550 –690– 850 2
2 20–50–80
16–20– 25 200 –340– 500
600 –750– 900 2
2 10–19–40 240– 265 –300 150 –300– 450
350 –485– 600 +
2 10–20–40 260– 285 –320 150 –275– 350
400 –460– 550 +
2 10–19–30 290– 325 –380 100 –200– 300
300 –405– 500 þ
2 20–30–45 110– 125 –160 150 –320– 400
350 –500– 650 2
2 15–26–35 110– 140 –160 450 –690– 950
650 –840– 950 2
+ 15–23–30 100– 115 –130 100 –300– 500
500 –605– 800 2
2 20–35–50 120– 140 –160 200 –350– 450
350 –460– 550 2
+ 15–22–30 120– 160 –190 300 –415– 550
500 –640– 800 2
+ 15–30–45 130– 145 –170 150 –240– 500
400 –495– 600 2
2
2
2
2
þ
2
þ
þ
þ
þ
þ
2
þ
2
2
2
2
2
2
þ
2
þ
þ
+
2
þ
2
2
2
+
+
2
2
+
2
+
2
+
+
2
2
+
2
2
+
+
2
2
2
+
+
+
2
2
2
2
2
+
2
þ
2
2
2
+
2
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
þ
þ
þ
+
+
+
2
2
+
þ
+
+
þ
þ
þ
+
+
+
þ
+
+
2
þ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
þ
C
þ
2
þ
2
þ
þ
þ
þ
þ
þ
2
2
þ
þ
þ
2
2
þ
2
þ
2
/
/
/
2– 14
/
2– 3
/
/
/
/
/
/
2
4– 6
/
/
/
2
2
/
2– 3
/
2
/
/
/
200 –2130–8000
/
300 –840– 1500
/
/
/
/
/
/
150 –345– 650
300 –550– 900
/
/
/
200 –300– 550
600 –950– 1800
/
200 –400– 700
/
300 –440– 600
þ
/
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
+
þ
þ
þ
þ
þ
þ
þ
þ
þ
P
P
S
S
S
S
S
S
P
Norm
Norm
Flat
Flat
Decr
Decr
Flat
Decr
Norm
+
+
2
2
2
2
+
+
+
þ
2
þ
þ
þ
þ
þ
þ
2
+
+
+
þ
þ
þ
þ
2
þ
2
2
2
2
2
2
2
2
2
+ 25– 55–100
þ 20–40–70
2 15–20–30
2 15–22–30
2 20–29–50
2 15–35–75
2 15–30–50
2 20–30–50
þ 25– 65–130
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
+
+
2
þ
þ
2
2
þ
þ
2
2
+
+
2
2
2
þ
þ
2
2
þ
þ
þ
+
2
2
2
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
þ
2
2
2
2
2
2– 3
3– 4
2– 3
/
2– 3
2– 8
2– 4
2– 3
2– 3
300 –565– 1000
250 –410– 600
100 –520– 1100
/
400 –760– 1850
600 –2060–4500
200 –720– 1600
600 –760– 1500
200 –400– 600
2
2
þ
þ
þ
þ
þ
þ
2
60–72– 85 300 –410– 550
600 –720– 850
60–75– 85 150 –330– 500 850– 1045–1200
140– 160 –180 300 –450– 600
400 –575– 700
210– 240 –280 450 –680– 1100
700 –840– 950
200– 215 –240 150 –285– 400
400 –525– 650
100– 135 –160 150 –270– 350
300 –380– 450
140– 180 –185 200 –390– 600
600 –725– 800
90– 105 –120 250 –450– 650
550 –680– 800
25–32– 38 300 –490– 650 800– 1020–1200
Tracheids thick-walled
Radial vessel multiples
þ
þ
þ
+
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Tracheid length (mm)
Solitary vessels
2
2
2
2
2
2
2
2
2
2
2
þ
+
2
þ
þ
+
2
2
2
2
2
+
Vessel element length (mm)
Tendency to (semi-)ringporosity
Flat
Decr
Flat
Flat
Decr
Decr
Decr
Decr
Flat
Decr
Flat
Decr
Flat
Flat
Flat
Flat
Flat
Flat
Flat
Flat
Flat
Decr
Decr
Vessel density (mm 2 1)
Length vs. age curve
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Vessel diameter (mm)
Primarily
or secondarily woody
Spermacoceae
Arcytophyllum lavarum*
Arcytophyllum setosum
Arcytophyllum thymifolium*
Diodella sarmentosa
Emmeorhiza umbellata*
Gomphocalyx herniarioides
Hedyotis flavescens*
Hedyotis fruticosa*
Hedyotis lessertiana*
‘Hedyotis’ trichoglossa
Kadua cordata
Lathraeocarpa acicularis
Mitracarpus frigidus
Nesohedyotis arborea 1
‘Oldenlandia’ ambovombensis 1*
‘Oldenlandia’ ambovombensis 2
‘Oldenlandia’ humbertii*
Spermacoceae bangweolensis
Spermacoce macrocephala
Spermacoce manikensis
Spermacoce occidentalis
Spermacoce verticillata 1
Spermacoce verticillata 2
Knoxieae
Carphalea kirondron*
Dirichletia virgata
Otiophora rupicola*
Otomeria micrantha
Pentanisia schweinfurtii 1
Pentanisia schweinfurtii 2*
Pentas zanzibarica
Phyllopentas schimperiana*
Triainolepis polyneura
2
2
2
2
2
2
2
2
2
1053
Dif, diffuse; Dia, diffuse-in-aggregates; P, primarily woody; S, secondarily woody; Decr, decreasing length vs. age curve; Flat, flat length vs. age curve; Norm, normal length vs. age curve; þ,
present; +, sometimes present; 2, absent; / indicates not applicable.
Lens et al. — Woodiness in Spermacoceae – Knoxieae
Species studied
TA B L E 2. Overview of selected wood anatomical characters within Spermacoceae and Knoxieae (Rubiaceae). Species are arranged alphabetically according to the
tribal studies of Groeninckx et al. (2009a) and Kårehed and Bremer (2007). Numbers between hyphens are mean values flanked by minimum and maximum values. For
specimens of the same taxon, superscript numbers after the species name refer to the order of the specimens as followed in the species list (see Appendix). Species
names in bold represent underground woody organs, and juvenile wood samples are marked with an asterisk behind the species names
1054
Lens et al. — Woodiness in Spermacoceae – Knoxieae
fibres from wood splinters taken at various distances between
the pith and cambium region – usually 1 mm between each
splinter – were measured using maceration slides. In
Table 2, the length of vessel elements and fibres were
always based on the outer part of the wood sample.
elaborate phylogenetic studies within these clades (Kårehed
and Bremer, 2007; Razafimandimbison et al., 2008).
R E S U LT S
Wood description
Phylogenetic analysis
The phylogenetic analysis is based on an existing data
matrix of atpB-rbcL, rps16, petD and trnL-trnF sequences,
which includes most of the Spermacoceae species studied
(Groeninckx et al., 2009a). The matrix was enlarged with
own sequences of Anthospermum sp., Coccocypselum decumbens, Colletecoema magna, Coprosma repens, Danais sp.,
Galium mollugo, Geophila repens, Mycetia malayana,
Myrmecodia tuberosa, Ophiorrhiza mungos, Paederia
foetida, Payera decaryi, Psychotria kirkii, Rubia fruticosa,
Saldinia sp., Serissa japonica, Spermacoce bangweolensis,
S. manikensis, Stelechantha makakana, Triainolepis polyneura
and Trichostachys aurea. Additional rps16 and trnL-trnF
sequences from GenBank were incorporated for Dirichletia
virgata (accession numbers AM266894, AM266980),
Pentanisia schweinfurthii (AM266861, AM266949), Pentas
zanzibarica (AM266892, AM266978) and Phyllopentas
schimperiana (AM266887, AM266973). No sequences were
obtained for the woody Spermacoceae Hedyotis flavescens,
Spermacoce macrocephala and S. occidentalis, and the
woody Knoxieae species Otomeria micrantha and Otiophora
rupicola. The combined matrix includes 133 taxa, of which
six basal Rubioideae were designated as the outgroup
(Coccocypselum
decumbens,
Colletecoema
magna,
Ophiorrhiza mungos, Saldinia sp., Stelechantha makakana
and Trichostachys aurea), eight Knoxieae species, 107 representatives of Spermacoceae and 12 species from additional
rubioid lineages. The dataset comprised 6045 characters, of
which 1021 were parsimony informative.
Parsimony analyses were conducted with NONA (Goloboff,
1999) using WinClada ver. 1.0000 as interface (Nixon, 2002).
A heuristic search was carried out with 1000 random addition
replicates,
tree-bisection
and
reconnection
(TBR)
branch-swapping holding ten trees per replicate, followed by
TBR branch-swapping on all trees resulting from the 1000
replicates. In order to evaluate the relative support of the
clades, jackknife (JS) and bootstrap (BS) analyses were executed using 1000 replicates with 100 initial trees holding one
tree per random addition, performing TBR to hold 1000
trees and calculating a consensus on each repetition.
Frequency values (.65%) were plotted onto the consensus
of the most-parsimonious trees.
The evolution of woodiness was investigated by deltran
optimization by adding four habit types [type 1 ¼ (sub)shrubs
(or occasionally treelets); type 2 ¼ woody herbs to (sub)shrubs; type 3 ¼ geoxylic herbs; type 4 ¼ herbs] onto the
strict consensus tree from the parsimony analysis using
WinClada ver. 1.0000 (Nixon, 2002). We chose deltran optimization to overcome the problem of restricted sampling in
several Rubioideae clades (such as, for example, Knoxieae
and Psychotrieae): the result was an optimization pattern that
agreed with what was expected based on the much more
The Spermacoceae studied are described according to the
recent tribal delimitation of Groeninckx et al. (2009a) and
Kårehed et al. (2008; Figs 2– 4 and 6A – D). For each genus
examined, the numerator presented in the text represents the
number of species studied and the denominator includes the
total number of species. Numbers without parentheses are
ranges of means, while numbers between parentheses represent
minimum or maximum values. Descriptions of continuous
characters are based on mature wood samples. A summary
of selected wood features is shown in Table 2. Because the
number of mature wood samples within Knoxieae is rather
limited, we decided not to provide a tribal wood description.
As an alternative, we have summarized our anatomical observations of Knoxieae in Table 2, added with illustrations (Figs 5
and 6E, F). The species of Spermacoceae that were studied and
their descriptions are as follows.
Arcytophyllum 3/17, Diodella 1/ 10, Emmeorhiza 1/1,
Gomphocalyx 1/1, Hedyotis 4/ 115, Kadua 2/28,
Lathraeocarpa 1/2, Mitracarpus 1/ 50, Nesohedyotis 1/1,
Oldenlandia 2/ 240, and Spermacoce 5/ 275 (Figs 2 – 4
and 6A– D). Growth ring boundaries usually distinct.
Diffuse-porous, but tendency to (semi-)ring-porosity in
Lathraeocarpa acicularis (Fig. 2C), Mitracarpus frigidus,
‘Oldenlandia’ ambovombensis, ‘Oldenlandia’ humbertii and
Spermacoce verticillata. Vessels (16) –20 – 540– (640) mm22,
mostly solitary and in radial multiples of 2 – 3 (Fig. 2A, B,
E), few additional vessel clusters in species of Diodella
(Fig. 2D), Lathraeocarpa (Fig. 2C) and Spermacoce; vessel
outline mostly slightly angular, sometimes rounded; perforation plates exclusively simple (Fig. 4D), sometimes a small
percentage of scalariform perforations in Hedyotis flavescens
(10%; 2– 5 bars) and H. fruticosa (20%; 4 – 10 bars), or a
high percentage of reticulate and irregular perforations in
Spermacoce bangweolensis (.50%). Intervessel pits alternate
(Fig. 4C), pits 3 – 5 – (6) mm in horizontal diameter, vestured;
additional ( pseudo-)scalariform vestured intervessel pits also
present in ‘Hedyotis’ trichoglossa (Fig. 4D), 10– 15 mm in
horizontal diameter. Vessel-ray pits similar to intervessel pits
in shape and size. Wall sculpturing absent. Tyloses sometimes
present in Gomphocalyx. Tangential diameter of vessels (10) –
16– 60– (120) mm, vessel elements (100) – 160 – 690– (1000)
mm long. Tracheids generally present, generally thin- or
thin-to-thick-walled but thick-walled in Lathraeocarpa and
Oldenlandia; pit borders generally distinctly bordered, (3)–
4 – 5 mm in diameter concentrated in tangential and radial
walls; tracheid length (150) – 200 – 1050– (1300) mm. Few
septate fibres with simple-to-minutely bordered pits ( pit
borders 2 – 3 mm in diameter and concentrated in radial
walls) found in Emmeorhiza, Hedyotis, Kadua, Mitracarpus
and Spermacoce; in ‘Hedyotis’ trichoglossa much more
septate fibres are present (Fig. 4C, D). Axial parenchyma
often scarce to apparently absent (Fig. 2B), usually a combination of diffuse or diffuse-in-aggregates apotracheal
Lens et al. — Woodiness in Spermacoceae – Knoxieae
A
B
C
D
1055
E
F I G . 2. Transverse light-microscope sections of woody Spermacoceae showing variation in vessel distribution, thickness of tracheids and scarcity of axial
parenchyma. (A) Arcytophyllum setosum. (B) Spermacoce macrocephala. (C) Lathraeocarpa acicularis, thick-walled tracheids and vessels in the ground
tissue. (D) Diodella sarmentosa, climbing species, wood cylinder partly broken up by large unlignified rays. (E) Nesohedyotis arborea, larger mean vessel diameter and lower vessel density than in most other woody Spermacoceae, axial parenchyma more common (arrows).
parenchyma with scanty paratracheal parenchyma (Fig. 2C),
parenchyma not observed in Arcytophyllum setosum
(Fig. 2A), Hedyotis lessertiana and ‘Hedyotis’ trichoglossa;
2 – 3 – (4) cells per parenchyma strand. Rays present, except
in Arcytophyllum setosum (Fig. 4A). Exclusively uniseriate
rays present in species of Arcytophyllum, Emmeorhiza,
Hedyotis, Kadua, Oldenlandia and Spermacoce (Fig. 4B);
0 – 25 rays mm21, (50)– 120 – 1950– (3300) mm long, consisting of upright cells (Fig. 4F). If present, multiseriate rays generally 2 – 3-seriate, 4– 6-seriate in Nesohedyotis arborea and
2 – 14-seriate in Diodella sarmentosa, (150) – 300– 2130 –
(8000) mm high, (0)– 2 – 5 rays mm21, consisting of mainly
upright or square ray cells (Fig. 4F), but an even distribution
of procumbent, square and upright ray cells in Nesohedyotis
arborea (Fig. 4E); sheath cells present in N. arborea.
No dark amorphous contents in ray cells. No crystals or
silica bodies observed.
Phylogenetic analysis
The phylogenetic analysis of the molecular data resulted in
8519 most-parsimonious trees with length 3412 (CI ¼ 0.49;
RI ¼ 0.75). The strict consensus tree with length 3511 (CI ¼
0.47; RI ¼ 0.74) is shown in Figure 7. The tribe Spermacoceae
s.l. was well supported, but the sister relationship with
Knoxieae was not found.
Within Spermacoceae s.l., a polytomy representing four
major lineages was present: (1) the herbaceous genera
Dentella – Pentodon; (2) a predominantly herbaceous lineage
comprising Kohautia and the Pentanopsis clade (including
1056
Lens et al. — Woodiness in Spermacoceae – Knoxieae
our analysis: Carphalea sister to Triainolepis, and the relationship between the genera Pentas, Batopedina and Pentanisia.
A
D IS C US S IO N
Diversity of wood anatomy within Spermacoceae
500 mm
B
500 mm
F I G . 3. Transverse light-microscope sections of the underground stem of
Spermacoce manikensis showing an initial wood cylinder that is more dramatically dispersed by intense divisions of the surrounding parenchyma tissue with
age. (A) Part of young stem, and (B) part of older stem.
the woody Lathraeocarpa and Gomphocalyx); (3) a lineage
including herbaceous Agathisanthemum species that are sister
to the woody herbs and (sub)shrubs of the Hedyotis s.s.
clade (including the type species H. fruticosa and
H. lessertiana); and (4) a large clade representing the remaining herbaceous and woody species of Spermacoceae. Within
this fourth clade, relationships were not fully resolved, but it
is clear that the woody species are scattered over different
clades, such as the Kadua clade, the Arcytophyllum –
Houstonia clade and the Spermacoceae s.s. clade. The
relationships within Knoxieae cannot be discussed in detail
because of the limited sampling. Nevertheless, some results
of the Kärehed and Bremer (2007) study were also found in
A typical feature in Spermacoceae wood is the flat or decreasing length vs. age curve for vessel elements (Fig. 6A – D).
Although we are aware that the maximal stem diameter in
some of our samples might not be wide enough to present
definitive length vs. age curves (e.g. Fig. 6B), we feel confident that the results are representative for the species. This is
predominantly based on the ideas of Bailey (1920), who
demonstrated that the vessel element length remains almost
constant with age in species having so-called ‘derived’
vessel elements, i.e. very short vessel elements (,350 mm)
combined with exclusively simple perforations. Furthermore,
Carlquist (1962) demonstrated that nearly all secondarily
woody species with very short vessel elements had a flat
length vs. age curve. Based on the maceration slides observed,
length vs. age curves for fibre-tracheids/tracheids are similar
but show less variation in length than vessel elements. For
example, average length values of fibre-tracheids/tracheids
from the pith towards the cambium range from 560– 495 mm
in Spermacoce verticillata (cf. Fig. 6A), from 500– 460 mm
in ‘Oldenlandia’ ambovombensis (cf. Fig. 6C) and from
925– 690 mm in Mitracarpus frigidus (cf. Fig. 6D).
Most Spermacoceae species observed had a similar wood
anatomy (Figs 2 and 4; Table 2). Besides the paedomorphic
length vs. age curves, the tribe could also be identified by
the following features: exclusively simple perforations, alternate and vestured intervessel pits, mainly solitary vessels in
combination with short radial multiples, narrow vessels
(usually 20– 40 mm in tangential width), high vessel densities
(often more than 100 mm22), relatively short vessel elements
and imperforate tracheary elements with distinctly bordered
pits (often between 150– 500 mm and 350 – 800 mm, respectively), scarce apo- and paratracheal axial parenchyma, and a
predominance of uniseriate rays including nearly exclusively
square-to-upright cells. Furthermore, about half of the
species studied had few septate libriform fibres, which most
likely act as a rapidly increased photosynthetic storage
capacity in woods with few (or nearly no) axial parenchyma
cells (so-called tracheid dimorphism, sensu Carlquist, 1988).
The evolution of fibre-tracheids towards septate libriform
fibres was most pronounced in the underground wood
sample of ‘Hedyotis’ trichoglossa, in which many tracheids
in the ground tissue have been replaced during evolution by
septate libriform fibres (Fig. 4C, D).
The variation in wood anatomy described above corresponds to the rubiaceous wood type I (Koek-Noorman, 1977;
Jansen et al., 2002), in which the type of imperforate
element in the ground tissue is the main characterizing
feature (I, tracheids/fibre-tracheids with distinctly bordered
pits vs. II, libriform fibres with simple-to-minutely bordered
pits). Furthermore, the fibre character in Rubiaceae was
found to be highly correlated with other wood characters,
such as vessel grouping (I, mainly solitary vs. II, mostly in
short multiples), axial parenchyma distribution (I, diffuse,
Lens et al. — Woodiness in Spermacoceae – Knoxieae
A
B
C
D
E
F
1057
F I G . 4. Tangential and radial longitudinal light-microscope sections of woody Spermacoceae showing intervessel pits (C, D) and ray characters. (A– C)
Tangential sections; (D– F) radial sections. (A) Arcytophyllum setosum, rayless wood. (B) Spermacoce macrocephala, abundance of uniseriate rays with
upright cells. (C) ‘Hedyotis’ trichoglossa, intervessel pits alternate (horizontal arrow), ground tissue mostly consisting of septate libriform fibres (vertical
arrows), intervessel pitting alternate. (D) ‘Hedyotis’ trichoglossa, simple perforation (vertical arrow), wide scalariform intervessel pitting (horizontal arrows).
(E) Nesohedyotis arborea, upper ray with many procumbent body ray cells (arrows). (F) Kadua cordata, ray consisting of upright cells.
diffuse-in-aggregates or banded vs. II, absent or scanty paratracheal) and ray width (I, narrow with long upright uniseriate
margins vs. II, wider rays with short uniseriate margins).
With regard to Spermacoceae, the combination of type I
fibres/tracheids with the axial parenchyma distribution
(scarce apotracheal and paratracheal parenchyma) and ray
structure (no difference between body and marginal ray
cells) is unusual within Rubiaceae, and can be at least partly
attributed to the secondary origin of the wood structure (see
following sections, below).
Some Spermacoceae species deserve special attention
because of their distinctive anatomy. The first one is
Diodella sarmentosa, the only straggling-to-climbing species
with mature wood in our study, which can be distinguished
by its wide and mainly unlignified rays (2 – 14-seriate) that
partly break up the wood cylinder (Fig. 2D). This division of
the wood cylinder is much more prominent in the underground
wood material of the geoxylic herb Spermacoce manikensis
(Fig. 3). Although the wood cylinder is more-or-less intact
in the youngest part of the underground structure (Fig. 3A),
it dramatically breaks down into several small ‘lignified
islands’ that float in a sea of dividing parenchyma cells
(Fig. 3B; see divided xylem cylinder according to the
cambial variant types of Carlquist, 2001). A similar, but less
pronounced cambial variant is encountered in the underground
structure of S. occidentalis, in which larger wood portions are
starting to get dispersed by parenchyma divisions. The abundant presence of parenchymatous tissue in these two species
1058
Lens et al. — Woodiness in Spermacoceae – Knoxieae
A
B
C
D
E
F
G
H
I
F I G . 5. Transverse (A– C), tangential (D– F) and radial (G– I) light-microscope sections of woody Knoxieae showing vessel distribution (A–C), ray width
(D– F) and ray composition (G–I) of primarily woody representatives (A, B, D, E, G, H) and secondarily woody members (C, F, I). (A, D, G) Dirichletia
virgata; (B, E, H) Triainolepis polyneura; (C, F, I) Pentas zanzibarica. (A) Tendency to dendritic vessel pattern; (B) pronounced radial vessel mutiples
ranging into dendritic pattern; (C) semi-ring porous wood, vessels mainly solitary and in short radial multiples; (D) body ray cells small and rounded; (E)
body ray cells small and rounded; (F) rays consisting of mainly upright ray cells; (G) body ray cells procumbent (arrows); (H) body ray cells procumbent,
enlarged ray cells containing raphides (arrows); and (I) multiseriate ray with mainly upright cells.
is probably related to an increased water-storage capacity in
order to cope with long periods of drought (Table 1).
Variation in wood anatomy within Knoxieae
From a wood-anatomy point of view, the variation within
Knoxieae is more complex than in Spermacoceae (Figs 5
and 6E, F). Primarily based on differences in the length vs.
age curve and the cellular composition of rays, the anatomical
diversity of wood in Knoxieae can be divided into two groups:
(1) Otomeria, Pentanisia, Pentas (Fig. 5C, F, I), and probably
Otiophora and Phyllopentas; and (2) Carphalea, Dirichletia
(Fig. 5A, D, G) and Triainolepis (Fig. 5B, E, H). The first
group of genera is nearly identical to the Spermacoceae
species studied: flat or decreasing length vs. age curves for
vessel elements (Fig. 6F, and to a lesser extent also for
fibre-tracheids/tracheids), exclusively simple perforations,
alternate and vestured intervessel pits, mainly solitary vessels
in combination with radial vessel multiples, narrow vessels
(usually 20– 50 mm in tangential width), high vessel densities
(often .100 mm22), relatively short vessel elements and
imperforate tracheary elements with distinctly bordered pits
(often between 200 – 600 mm and 450– 800 mm, respectively),
and a predominance of upright-to-square ray cells in uni- and
multiseriate rays (Fig. 5F, I). More mature material of
Otiophora and Phyllopentas needs to be carefully studied in
order to investigate specific characters more in detail, such
as length vs. age curves and cellular ray composition, before
Lens et al. — Woodiness in Spermacoceae – Knoxieae
600
A Spermacoce verticillata
Vessel element length (mm)
Vessel element length (mm)
700
600
500
400
300
200
100
B Lathraeocarpa acicularis
500
400
300
200
100
0
0
0
1000
2000
3000
4000
5000
6000
0
200
400
Distance from pith (mm)
800 1000 1200 1400 1600 1800 2000
Distance from pith (mm)
C ‘Oldenlandia’ ambovombensis
D Mitracarpus frigidus
Vessel element length (mm)
Vessel element length (mm)
600
1400
600
500
400
300
200
100
0
1200
1000
800
600
400
200
0
0
500 1000 1500 2000 2500 3000 3500 4000 4500 5000
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Distance from pith (mm)
900
Distance from pith (mm)
700
E Triainolepis sp.
800
Vessel element length (mm)
Vessel element length (mm)
1059
700
600
500
400
300
200
F Pentas zanzibarica
600
500
400
300
200
100
100
0
0
1000
2000
3000
4000
5000
6000
7000
Distance from pith (mm)
0
0
500
1000
1500
2000
2500
3000
3500
Distance from pith (mm)
F I G . 6. Length vs. age curves for vessel elements in Spermacoceae (A– D) and Knoxieae (E– F). Vessel element length strongly decreases in the primary xylem
and the first-formed wood (close to the pith), followed by a subsequent slight decrease (decreasing curve, A, B) or subsequent stabilization (flat curve, C, D, F) or
slight increase (normal curve, E) towards the cambium. (A– D, F) Secondarily woody species; (E) primarily woody species.
we can more confidently place these two genera in the first
group.
The second group of species can be distinguished from the
first group by the presence of normal length vs. age curves
(Fig. 6E) and the predominance of procumbent body ray
cells (Fig. 5D, E, G, H). Among the three species of the
second group, Dirichletia virgata and Triainolepis polyneura
are remarkable in having prominent radial vessel multiples
and/or large vessel clusters (even a strong tendency to a dendritic vessel pattern in D. virgata; Fig. 5A), wider vessels
(up to 130 mm in tangential width), and much longer fibres
(800 – 1200 mm; Table 2). In addition, the juvenile sample of
1060
Lens et al. — Woodiness in Spermacoceae – Knoxieae
Carphalea kirondron also showed a tendency to produce more
pronounced vessel multiples and wider vessels. The difference
in vessel width and fibre length between both groups can be
related to differences in habit: species of the second group
are all shrubs to trees, which are taller than the woody
members of the first group (smaller shrubs to woody herbs
or geoxylic herbs; Verdcourt, 1976). In addition, the pronounced vessel multiples in Dirichletia and Triainolepis
provide support for their adaptation to relatively dry (semi-)
deciduous forests (Carlquist, 1984; Table 1).
When the diversity of wood anatomy of Knoxieae is compared
with the remaining Rubiaceae, it is evident that the anatomical
characters of the three species of the second group (i.e.
Triainolepis polyneura, Dirichletia virgata and Carphalea kirondron) fit better within the rubiaceous wood type I due to their
narrow rays with long uniseriate ends (Fig. 5D, E). However,
the pronounced vessel multiples, especially in Triainolepis and
Dirichletia (Fig. 5A-B), strikingly contradict the general occurrence of solitary vessels in type I Rubiaceae (Jansen et al., 2002).
Are Spermacoceae and Knoxieae primarily woody or secondarily
woody?
With respect to the wood anatomy of Spermacoceae, the presence of flat or decreasing length vs. age curves for vessel
elements (and to a lesser extent also fibre-tracheids/tracheids)
in all the species observed (Fig. 6A – D) and the occurrence of
mainly square-to-upright ray cells in nearly all the species
observed [Fig. 4B– D, F; rays absent in Arcytophyllum
setosum (Figs 2A and 4A); deviating ray cell morphology in
Nesohedyotis arborea] provide strong arguments for secondary
woodiness (Carlquist, 1962, 1992). Our study also provides new
evidence that these typical paedomorphic features are also valid
in underground woody organs of geoxylic herbs. In addition, the
presence of wide scalariform intervessel pits in combination
with the normal alternate vessel pits in ‘Hedyotis’ trichoglossa
provides further support for the general validity of paedomorphic features in underground woody structures (Fig. 4C,
D). Nevertheless, there are some wood features that deviate
from the typical paedomorphic wood pattern. For instance,
rays with a significant percentage of procumbent body ray
cells were observed throughout the large wood sample of the
only arborescent member within the tribe Spermacoceae that
we investigated, Nesohedyotis arborea (trees up to 7 m tall).
Apparently, the expanded ontogeny of wood cells in this
species has changed many body ray cells into the matured procumbent shape (Fig. 4E). Nevertheless, according to the overview study of Carlquist (1962), it is the gradual decrease in
vessel element length towards the outer stem parts that is the
decisive factor to infer secondary woodiness in a given
species [although the pith region was not included in our large
sample (70 mm in diameter), we did measure, on average, a
150-mm decrease in vessel element length between the youngest
part and the cambium zone]. Other wood features in
Spermacoceae that are unusual in paedomorphic species were
the low level of parenchymatization throughout the tribe
(Fig. 2A –C; except in the underground structure of
Spermacoce manikensis, Fig. 3) and the presence of thickwalled fibres/tracheids in the genera Oldenlandia and
Lathraeocarpa (Carlquist 1962, Fig. 2C).
According to our observations of wood anatomy, the origin
of woodiness in Knoxieae is more complex. All taxa observed
of the first group (Otiophora, Otomeria, Pentanisia, Pentas
and Phyllopentas) are hypothesized to be secondarily woody
based on the presence of paedomorphic length vs. age
curves for vessel elements (and to a lesser extent also fibretracheids/tracheids) and the predominance of square-to-upright
ray cells (Figs 5C, F, I and 6F). In contrast, the three genera
observed in the second group (Carphalea, Dirichletia and
Triainolepis) can be distinguished from the first group by
wood features that are typical of ‘normal’ primarily woody
dicots: (1) length vs. age curves indicate a strong initial
decrease of vessel element length in the primary xylem and
in the first-formed wood, followed by an increase and subsequent stabilization (Fig. 6E), and (2) rays are composed of
procumbent body ray cells and square-to-upright rows of marginal ray cells (Fig. 5D, E, G, H).
Figure 7 demonstrates that the general assumption of a primarily woody Rubioideae ancestor is plausible: early-diverging
Rubioideae clades include many woody shrubs or (small) trees
having non-paedomorphic rays with procumbent body ray cells
and upright marginal ray cells, such as for example
Stelechantha (Urophylleae; Jansen et al., 2001), Lasianthus
(Lasiantheae, represented by Saldinia and Trichostachys in
Fig. 7; Jansen et al., 2001) and Psychotria (Psychotrieae;
Jansen et al., 1997). Moreover, our study in Knoxieae reveals
that at least some primarily woody species remain present in
deeply nested Rubioideae lineages. With respect to Knoxieae,
the molecular phylogeny of Kårehed and Bremer (2007) shows
that our three primarily woody species are all placed in earlydiverging branches within the tribe, which would indicate that
the ancestor of Knoxieae was probably primarily woody
(Fig. 7). This is further corroborated by the fact that all species
of Carphalea, Dirichletia and Triainolepis are clearly woody
(shrubs to small trees), although the earliest diverging
Knoxieae lineage, Chamaepentas, consists of species that are
considered to be woody herbs (Verdcourt, 1976). In contrast,
all the secondarily woody species studied belong to laterdiverging Knoxieae branches (Kårehed and Bremer, 2007), in
which entirely herbaceous or herbaceous-like shrubby species
are common (Verdcourt, 1976). Within Spermacoceae s.l.,
Fig. 7 demonstrates that woodiness has developed multiple
times in all major essentially herbaceous lineages, and therefore
current molecular evidence suggests that the ancestor of
Spermacoceae was herbaceous. As a result, woody members
are best interpreted as secondarily woody.
Consequently, the present molecular phylogenies seem to
support our wood anatomical hypothesis that points to
primary as well as secondary woodiness in Knoxieae on the
one hand, and to exclusively secondary woodiness within
Spermacoceae on the other. However, it should be kept in
mind that several important nodes within the molecular phylogeny of Rubioideae as a whole and Spermacoceae – Knoxieae
in particular are not fully resolved.
Ecology of secondarily woody species
Why do some species that belong to essentially herbaceous
clades develop a woody habit? First of all it should be noted
that probably a majority of Spermacoceae species are not
Lens et al. — Woodiness in Spermacoceae – Knoxieae
100/100
Colletoecema magna
[continued, left]
Stelechantha makakana
Ophiorrhiza mungos
(Sub)shrubs(occasionally treelets)
Saldinia sp.
100/100
Woody herbs to (sub)shrubs
Trichostachys aurea
84/88
Geoxylic herbs
Coccocypselum decumbens
89/88
Herbs
Geophila repens
100/100
82/85
Psychotria kirkii
83/86
100/100
98/97
Myrmecodia tuberosa
89/91
Danais sp.
Payera decaryi
65/67
99/100
Coprosma repens
99/100
100/100
Anthospermum sp.
91/95
Mycetia malayana
94/95
Paederia foetida
98/99
Serissa japonica
91/94
Rubia fruticosa
97/96
100/100
Galium mollugo
70/75
Dirichletia virgata*
Phyllopentas schimperiana*
100/100
Carphalea madagascariensis
Triainolepis sp.*
99/99
Pentas zanzibarica*
Batopedina pulvinellata
99/99
Pentanisia parviflora
99/100
Knoxieae
Pentanisia Schweinfurthii*
100/100
Dentella repens
100/100
Pentodon pentandrus
Agathisanthemum globosum
100/100
100/100
98/98
Agathisanthemum bojeri
99/100
Lelya osteocarpa
Oldenlandia
uniflora
98/98
98/99
Oldenlandia angolensis
83/86
Oldenlandia goreensis
Hedyotis fruticosa*
99/99
Hedyotis korrorensis
99/99
Hedyotis lawsoniae
Hedyotis rhinophylla
99/100
Hedyotis lessertiana*
Hedyotis quinquenervis
Kohautia cynanchica
100/100
99/100
94/97
Kohautia caespitosa
Kohautia coccinea
Manostachya ternifolia
81/88
Oldenlandia rosulata
91/95
Lathraeocarpa
acicularis*
100/100
97/99
Gomphocalyx herniarioides*
99/99
Phylohydrax madagascariensis
Conostomium quadrangulare
100/100
Oldenlandia hebacea
100/100
83/89
Amphiasma luzuloides
100/100
Oldenlandia affinis
100/100
Spermacoceae s.l.
Pentanopsis fragrans
Dibrachionostylus kaessneri
Oldenlandia echinulosa
Oldenlandia geophila
Spermacoceae s.s.
Oldenlandia fastigiata
100/100
Hedythyrsus spermacocinus
100/100
Mitrasacmopsis quadrivalvis
Oldenlandia mitrasacmoides
Oldenlandia nervosa
97/97
Synaptantha tillaeacea
Oldenlandia tenelliflora
93/94
68/66
Oldenlandia galioides
Oldenlandia lancifolia
Oldenlandia biflora
Kadua acuminata
96/93
Kadua coriacea
Kadua rapensis
76/76
Kadua centranthoides
76/77
Kadua foggiana
85/85
Kadua cordata*
96/98
Kadua elatior
82/81
Kadua littoralis
Kadua degeneri
Kadua parvula
[continued, right]
1061
Astiella delicatula
“Hedyotis” trichoglossa*
“Oldenlandia” ambovombensis*
“Oldenlandia” humbertii*
Oldenlandia mircotheca
Stenaria nigricans
91/97
Houstonia caerulea
Houstonia longifolia
Arcytophyllum thymifolium*
Arcytophyllum lavarum*
Arcytophyllum muticum
73/75
Arcytophyllum rivetii
Arcytophyllum setosum*
Kohautia microcala
Kohautia obtusiloba
Kohautia virgata
Oldenlandia wiedemannii
Oldenlandia capensis
95/95
Oldenlandia nematocaulis
85/85
64/70
Oldenlandia robinsonii
Oldenlandia corymbosa
Oldenlandia taborensis
Oldenlandia wauensis
Nesohedyotis arbotea*
Bouvardia glaberrima
Bouvardia ternifolia
Manettia alba
Manettia lygistum
Oldenlandia salzmannii
Oldenlandia tenuis
Emmeorhiza umbellata*
Spermacoce verticillata*
Crusea megalocarpa
Crusea calocephala
Spermacoce filifolia
Spermacoce flagelliformis
Spermacoce Prostrata
Spermacoce erosa
86/89
Spermacoce marginata
Richardia scabra
Richardia stellaris
Galianthe eupatorioides
97/97
85/83
Diodia spicata
Galianthe brasiliensis
Spermacoce capitata
68/74
70/76
Hemidiodia ocymifolia
Spermacoce remota
97/97
Mitracarpus frigidus*
Mitracarpus microspermus
Diodella sarmentosa*
Diodia aulacosperma
Ernodea littoralis
Spermacoce bangweolensis*
Spermacoce filituba
Spermacoce hispida
Spermacoce manikensis*
Spermacoce ruelliae
F I G . 7. Strict consensus tree based on maximum-parsimony analysis of atpB-rbcL, rps16, petD and trnL-trnF showing bootstrap/jackknife support values above
the branches. The colour of the species’ names indicates their habit: black ¼ (sub)shrubs (or occasionally treelets); brown ¼ woody herbs to (sub)shrubs;
purple ¼ geoxylic herbs; green ¼ herbs. Asterisks indicate the species of woody Spermacoceae and Knoxieae taxa that were investigated in this study. This
tree supports our wood anatomical hypothesis: the common ancestor of Knoxieae is primarily woody, while the common ancestor of Spermacoceae is herbaceous.
annuals but short-lived perennials. In addition, some essentially annual species become short-lived perennials when
growth conditions (ideal temperature and water availability)
allow the species to grow longer. The wood production in
most of these species, however, is limited. Geoxylic herbs,
sometimes developing considerable amounts of secondary
derived wood (Fig. 1A, detail), probably evolved to withstand
longer periods of drought. This could, for example, be true for
1062
Lens et al. — Woodiness in Spermacoceae – Knoxieae
Spermacoce manikensis growing on the nutrient poor Kalahari
sands, which are inundated during the rainy season but are
completely dry for the rest of the year. In this species there
is also evidence that the massive amount of parenchyma
tissue in the underground organs plays a role in water
storage (Fig. 3B). Another evolutionary advantage of geoxylic
herbs is their ability to survive under periodic fire conditions,
which has been clearly demonstrated for example in some
Otiophora species (Knoxieae; Puff, 1981). Other secondarily
woody Spermacoceae – Knoxieae can be generally described
as woody herbs, subshrubs or shrubs, and some grow in habitats that are dry for at least part of the year (Table 1); in particular, the woody Spermacoceae growing in Madagascar
undergo severe drought stress (Table 1). Consequently, it
appears that – at least for some Spermacoceae and Knoxieae
species – recurring water stress goes hand-in-hand with
wood production.
From the point of view of wood anatomy, there are some
well-known features that can be correlated with dry habitats,
such as exclusively simple vessel perforations, vestured intervessel pits, high vessel densities (often more than 100
mm22), narrow vessels (often between 20– 40 mm), short
vessel elements (often below 350 mm, although this is also
induced by the secondary woodiness), and the presence of
water-conducting tracheids in the ground tissue that acts as a
subsidiary water transport system in case the vessels become
embolized (Carlquist, 1966, 1984; Baas et al., 1983;
Carlquist and Hoekman, 1985; Baas, 1986; Choat et al.,
2004). The species observed that have the most pronounced
xeromorphic wood features were Arcytophyllum thymifolium,
Lathraeocarpa acicularis, ‘Oldenlandia’ ambovombensis and
‘Oldenlandia’ humbertii. Furthermore, the latter three
species were the only ones having thick-walled (narrow)
vessels and thick-walled fibre-tracheids/tracheids in their
ground tissue, which were linked in order to support against
vessel implosion by negative pressures in the xylem during
drought (Hacke et al., 2001; Jacobsen et al., 2005). Not surprisingly, these three species are found in the markedly dry
south-western part of Madagascar, characterized by a short
rain season and a long dry season (Jury, 2003; Table 1).
Although the link between drought resistance and wood production is also observed in other angiosperms, such as some
additional Rubiaceae (Rubia and Crucianella; Koek-Noorman,
1976), Brassicaceae (Carlquist, 1971), and some Asteraceae
(Argyroxiphium and Dubautia; Carlquist, 2003), it is certainly
not the most typical habitat for secondarily woody species to
occur. Indeed, Table 1 shows that most non-Malagasy
Spermacoceae/Knoxieae species studied have developed a secondarily woody habitat in areas that do not experience severe
drought stress (cf. Carlquist, 1974). Examples are oceanic
islands such as Hawaii (Kadua cordata) and St. Helena
(Nesohedyotis arborea), and various high-elevation tropical
montane areas (Arcytophyllum, Hedyotis). Likewise, when we
look for a worldwide distribution of secondarily woody species
throughout angiosperms, it is evident that environments with
uniform annual temperatures and rainfall (for instance, on
oceanic islands and tropical mountains) seem perfectly suitable
for secondary woodiness to occur (Carlquist, 1974).
The degree of woodiness within most species studied
(woody herbs or shrubs) appears to be related to the
temperature and amount of rainfall. For example, the particular
environmental conditions on St. Helena (15 – 208C mean
annual temperature, approx. 1000 mm annual precipitation)
favour the arborescent habit of Nesohedyotis arborescens,
and might explain why this species is the only truly arborescent Spermacoceae in our study. This aberrant habit type
also accounts for the wider and fewer vessels of
N. arborescens (Fig. 2E) compared to the other smaller
woody species studied, although vessel element length is comparable (Table 2).
The numerous independent evolutionary shifts from herbaceousness to secondary woodiness in various flowering plant
families and in diverse habitats must go hand-in-hand with a
flexible genetic background mechanism that allows these frequent shifts. Preliminary evidence for this plastic genetic
basis is provided by Melzer et al. (2008), who demonstrated
that the inactivation of only two genes in the herbaceous
Arabidopsis thaliana resulted in a perennial-like, shrubby
mutant phenotype.
ACK N OW L E DG E M E N T S
We thank Dr Sherwin Carlquist and Dr Jesper Kårehed for
their valuable discussions, and the director of the BR herbarium and the curators of the xylaria of Kew (Kw), Tervuren
(Tw) and Utrecht (Uw) for their supply of wood samples.
We also thank Nathalie Geerts for technical assistance. This
work was supported by research grants of the K.U. Leuven
(grant number OT/05/35) and the Fund for Scientific
Research – Flanders (Belgium) (F.W.O. – Vlaanderen)
(grant number G.0250.05). F.L. is postdoctoral fellow of the
Fund for Scientific Research – Flanders (Belgium) (F.W.O. –
Vlaanderen). I.G. holds a PhD grant from the F.W.O. –
Vlaanderen.
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APPENDIX
List of taxa investigated in this study with reference to their
locality, vouchers and stem diameter. Nomenclature of
species’ names follow Govaerts et al. (2006) and Kårehed
and Bremer (2007). Institutional wood collections used in
this study are abbreviated according to Index Xylariorum
(Stern, 1988). Species names in bold refer to underground
wood material.
SPERMACOCEAE: Arcytophyllum lavarum K.Schum:
Costa Rica, J. Jangoux 1394 (BR), 3 mm; Arcytophyllum
setosum (Ruiz & Pav.) Schltdl.: Ecuador, P. M. Jørgensen
et al. 2378 (BR), 6 mm; Arcytophyllum thymifolium (Ruiz &
Pav.) Standl.: Ecuador, F. Billiet and B. Jadin 6580 (BR),
3 mm; Diodella sarmentosa (Sw.) Bacigalupo & E.L.Cabral
ex Borhidi: Cameroon, S. Dessein & B. Sonké 1423 (BR),
8 mm; Emmeorhiza umbellata (Spreng.) K.Schum: origin
and collector unknown (BR-S.P.802 367), 3.5 mm;
Gomphocalyxherniarioides Baker: Madagascar, P. De Block
2217 (BR), 4 mm; Hedyotis flavescens Thwaites: Sri Lanka,
Fosberg 58004 (BR), 5 mm; Hedyotis fruticosa L.: Sri
Lanka, D. D. Tirvangadum 663 (BR), 4 mm; Hedyotis lessertiana Arn.: Sri Lanka, P. L. Comanor 959 (BR), 4 mm;
‘Hedyotis’
trichoglossa
Baker
(unplaced
taxon):
Madagascar, H. Humbert 17722 (BR), 7 mm; Kadua cordata
Cham. & Schltdl.: Hawaii Islands (USA), Stern, W.L. 2996
(Uw 18607), 7 mm; Lathraeocarpa acicularis Bremek.:
Madagascar, P. De Block 2316 (BR), 6 mm; Mitracarpus
1064
Lens et al. — Woodiness in Spermacoceae – Knoxieae
frigidus (Willd. ex Roem. & Schult.) K.Schum.: National
Botanic Garden of Belgium, F. Van Caekenberghe 2 (BR
living collection), 19 mm; Nesohedyotis arborea (Roxb.)
Bremek.: UK (St. Helena), J. C. Melliss s.n. (Kw 11160),
mature (about 70 mm); ‘Oldenlandia’ ambovombensis sp.
nov. ined.: Madagascar, P. De Block 2328 (BR), 4 mm,
11 mm; ‘Oldenlandia’ humbertii sp. nov. ined.: Madagascar,
P. De Block 2294 (BR), 3 mm; Spermacoce bangweolensis
(R.E.Fr.) Verdc.: Zambia, Dessein et al. 550 (BR), 20 mm;
Spermacoce manikensis Dessein: Zambia, Dessein et al. 976
(BR), 16 mm; Spermacoce macrocephala (Standl. &
Steyerm.) Govaerts: Venezuela, B. Maguire et al. 41668 (Tw
36238), 7 mm; Spermacoce occidentalis Hardwood:
Australia, Harwood 1541 (BR), 10 mm; Spermacoce verticillata L.: South America, Cavalcanti et al. 358 (Uw 33690),
10 mm; Spermacoce verticillata L: Cultivated in the
National Botanic Garden of Belgium, F. Van Caekenberghe
104 (BR), 11 mm;
KNOXIEAE: Carphalea kirondron Baill. subsp. geayi
(Homolle) Puff: Madagascar, A. M. Homolle 1415 (BR),
6 mm; Dirichletia virgata (Balf.f.) Kårehed & B.Bremer:
Yemen (Socotra Archipelago), Schweinfurth 43 (Uw 25955),
mature; Otiophorarupicola Verdc., Burundi, M. Reekmans
178 (BR), 7 mm; Otomeria micrantha K.Schum.: DR Congo,
J. Louis 3389 (BR), 5 mm; Pentanisia schweinfurthii Hiern:
DR Congo, G. F. de Witte 07197 (BR), 7 mm; Pentanisia
schweinfurthii Hiern: DR Congo, G. F. de Witte 02767
(BR), 5 mm; Pentas zanzibarica Vatke var. rubra Verdc.:
DR Congo, Lebrun 3691 (BR), 8 mm; Phyllopentas schimperiana (Vatke) Kårehed & B.Bremer: Ethiopia, C. Puff 810920 –
1/2 (Uw 27081), 8 mm; Triainolepis polyneura Bremek.:
Madagascar, P. De Block 593 (BR), 16 mm.