Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae)

Abstract

The little known Rubiaceae genus Villaria is endemic mostly to the coastal forests of the Philippines. Traditionally, it has been placed in the tribe Gardenieae. Later
it was transferred to Octotropideae sensu Robbrecht and Puff. Villaria was placed among the “primitive” genera of the tribe, which are essentially characterized by large fruits, horizontal ovules
and numerous seeds. Parsimony and Bayesian analyses of the combined plastid (rps16 and trnT-F) dataset strongly support the inclusion of Villaria in Octotropideae as well as monophyly of the genus. However, our molecular results do not conform to the current informal
groups of the tribe delimited by fruit size, ovule position, number of seeds and exotesta thickenings. Instead, a close relationship
between Villaria and two “central genera” (Hypobathrum and Pouchetia) is revealed for the first time. This clade is sister to a group comprising “primitive” (Fernelia), “advanced” (Kraussia and Polysphaeria) and “central” (Feretia) representatives. In addition, our combined tree strongly supports a sister taxa relationship between Canephora and Paragenipa. Villaria is characterized by unilocular ovaries, parietal placentation and strictly horizontal ovules. These features are unique within
the Octotropideae. We recognize a total of five Villaria species, one new species (V. leytensis) is described here, and two species (V. philippinensis and V. rolfei) are transferred into synonymy with V. odorata. Each species is fully described, and a key to the species, a distribution map and illustrations are provided.

KeywordsIxoroideae–Octotropideae–Philippines–
rps16 intron–Rubiaceae–
trnT-F region–
Villaria

Full text

1 23
Plant Systematics and Evolution
ISSN 0378-2697
Volume 296
Combined 1-2
Plant Syst Evol (2011) 296:1-20
DOI 10.1007/s00606-011-0472-9
Molecular phylogeny and taxonomic
revision of the Philippine endemic Villaria
Rolfe (Rubiaceae)
Grecebio Jonathan D. Alejandro, Ulrich
Meve, Arnaud Mouly, Mike Thiv & Sigrid
Liede-Schumann

1 23
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ORIGINAL ARTICLE
Molecular phylogeny and taxonomic revision of the Philippine
endemic Villaria Rolfe (Rubiaceae)
Grecebio Jonathan D. Alejandro
•
Ulrich Meve
•
Arnaud Mouly
•
Mike Thiv
•
Sigrid Liede-Schumann
Received: 26 October 2010 / Accepted: 5 May 2011 / Published online: 31 May 2011
! Springer-Verlag 2011
Abstract The little known Rubiaceae genus Villaria is
endemic mostly to the coastal forests of the Philippines.
Traditionally, it has been placed in the tribe Gardenieae.
Later it was transferred to Octotropideae sensu Robbrecht
and Puff. Villaria was placed among the ‘‘primitive’’
genera of the tribe, which are essentially characterized by
large fruits, horizontal ovules and numerous seeds. Parsi-
mony and Bayesian analyses of the combined plastid
(rps16 and trnT-F) dataset strongly support the inclusion of
Villaria in Octotropideae as well as monophyly of the
genus. However, our molecular results do not conform to
the current informal groups of the tribe delimited by fruit
size, ovule position, number of seeds and exotesta thick-
enings. Instead, a close relationship between Villaria and
two ‘‘central genera’’ (Hypobathrum and Pouchetia) is
revealed for the first time. This clade is sister to a group
comprising ‘‘primitive’’ (Fernelia), ‘‘advanced’’ (Kraussia
and Polysphaeria) and ‘‘central’’ (Feretia) represe ntatives.
In addition, our combine d tree strongly supports a sister
taxa relationship between Canephora and Paragenipa.
Villaria is characterized by unilocular ovaries, parietal
placentation and strictly horizontal ovules. These features
are unique within the Octotropidea e. We recognize a total
of five Villaria species, one new species (V. leytensis) is
described here, and two species (V. philippinensis and V.
rolfei) are transferred into synonymy with V. odorata. Each
species is fully described, and a key to the species, a dis-
tribution map and illustrations are provided.
Keywords Ixoroideae ! Octotropideae ! Philippines !
rps16 intron ! Rubiaceae ! trnT-F region ! Villaria
Introduction
Villaria Rolfe is a little known endemic Rubiaceae genus
of the Philippines. It was established by Rolfe (1884) in
commemoration of Fr. Celestino Fernandez-Villar, a
Spanish priest and botanist, who processed and revised the
3rd edition of Blanco’s Flora Filipina (1877–1883). Before
the present study, six Villaria species, ranging from shrubs
to small-sized trees, were recognized: V. acutifolia (Elmer)
Merr., V. fasciculiflora Quisumb. & Merr., V. glomerata
(Bartl. ex DC.) Mulyan. & Ridsdale, V. odorata (Blanco)
Merr., V. philippine nsis Rolfe and V. rolfei S. Vidal.
According to the protologue, Villaria is characterized
within Rubiaceae by the combined possession of cymose,
axillary inflorescences, contorted corolla aestivation, uni-
locular ovaries, biparietal placentas, horizontal ovules and
G.J.D. Alejandro dedicates this work to the late Eduardo P. de Leon, a
good friend who made several collections of Villaria.
G. J. D. Alejandro (&)
College of Science and Research Center for the Natural
Sciences, University of Santo Tomas, Espan
˜
a Boulevard,
1015 Manila, Philippines
e-mail: gdalejandro@mnl.ust.edu.ph; balejan@yahoo.com
G. J. D. Alejandro ! U. Meve ! S. Liede-Schumann
Department of Plant Systematics, University of Bayreuth,
Universita
¨
tstr. 30, 95440 Bayreuth, Germany
A. Mouly
CNRS UMR 6249 Chrono-environnement, UFR Sciences et
Techniques, Universite
´
de Franche-Comte
´
,
16 Route de Gray, 25030 Besanc¸on cedex, France
M. Thiv
Botany Department, Museum of Natural History Stuttgart,
Rosenstein 1, 70191 Stuttgart, Germany
123
Plant Syst Evol (2011) 296:1–20
DOI 10.1007/s00606-011-0472-9
Author's personal copy

densely indumented stigmas. Other significant characters to
fully set apart the genus (e.g. radicle position, pollen
morphology, seed morphology) are still wanting.
Rolfe (1884) was certain of a close relationship between
Villaria and the widely distributed large genus Gardenia
Ellis because they share a unilocular ovary, a rare feature in
the coffee family. Subsequently, Rubiaceae specialists (e.g.
Schumann 1891; Verdcourt 1958; Bremekamp 1966)
placed Villaria in the tribe Gardenieae. Later, Robbrecht
(1980) transferred some genera with superior embryo
radicles and axillary inflorescences from the largely het-
erogeneous Gardenieae to the palaeotropical Hypobathreae
(Miq.) Robbr. The former two subtribes of Hypobathreae
(Hypobathrinae and Lamprothamninae), characterized by
entire endosperm and numerous ovules vs. ruminate
endosperm and few ovules, were replaced by three infor-
mal groups: ‘‘primitive’’, ‘‘central’’ and ‘‘advanced’’
(Robbrecht and Puff 1986). They included Villaria among
the ‘‘primitive’’ genera characterized by large fruits
developing numerous seeds from horizontal ovules. Hyp-
obathreae was later rena med as Octotropideae Beddome
when the South Indian Octotropis Beddome was included
within this tribe (Robbrecht et al. 1993 ).
Currently, Octotropideae comprise about 30 genera
distributed in the Old World and almost all known mem-
bers possess bilocular ovaries with axile placentation
(Robbrecht 1988; Bridson and Verdcourt 2003; Ruhsam
and Davis 2007). A close relationship between Cremaspora
Benth. and Octotropideae is consistent in several recent
phylogenetic studies (Andreasen and Bremer 2000; Persson
2000; Robbrecht and Manen 2006; Davis et al. 2007; Tosh
et al. 2008; Bremer and Eriksson 2009), but the base of the
clade they form is usually not resolved and supported. The
circumscription of Octotropideae is consequently not
clearly settled. Robbrecht and Manen (2006) recommended
inclusion of Cremaspora in Octotropideae, a suggestion
followed in the present work, pending further indications.
The position of Villaria in the tribe remains obscure due to
its rather unique unilocular ovary with parietal placenta-
tion. To date, a comprehensive study including a phylog-
eny of Octotropideae is lacking. Available studies
constitute not even half of the members to completely
understand the tribe. The majority of these revisions
include Madagascan Flagenium Baill. (Ruhsam and Davis
2007), Gallienia Dubarb & Dop (Dubard and Dop 1925;
Stone and Davis 2004), Jovetia Gue
´
de
`
s (Leroy 1976
) and
Lemyrea (A. Chev.) A. Chev. & Beille (Beille 1939; Stone
and Davis 2004), followed by African Petitiocodon Robbr.
(Tosh et al. 2008), Polysphaeria Hook.f. (Verdcourt 1988),
Pouchetia A. Rich ex DC. (Robbrecht et al. 1991); Masc-
arene Ramosmania Tirveng. & Verdc. (Tirvengadum and
Verdcourt 1989), Fernelia Comm. ex Lam. (Verdcourt
1989), Seychelles Paragenipa Baill. (Tirvengadum and
Robbrecht 1985), and only two Asian representatives
Nargedia Beddome (Tirvengadum and Robbrecht 1985)
and Octotropis (Robbrecht et al. 1993). Therefore, our
study represents a further step towards the understanding of
Octotropideae. Molecular data of Villaria from two plastid
regions (rps16 and trnT-F) are incorporated along with
previously published plastid sequences of Ixoroideae
(e.g. Andersson and Rova 1999; Persson 2000, Rova
et al. 2002) in order to test the validity of its phyloge-
netic placement in Gardenieae as previo usly circum-
scribed, or whether it should be placed in the informal
groups within Octotropideae recognized by Robbrecht
and Manen (2006).
Species delimitation within Villaria is currently based
on overlapping morphological traits. One year after Vil-
laria and its type species V. philippinensis were estab-
lished, Vidal (1885) described two other species, V.
littoralis and V. rolfei. Although Vidal observed close
similarities between the former and V. philippinensis, and
between the latter and V. littoralis, he recognized the three
elements as separate species and constructed a superficial
key mainly based on leaf size and shape. Later, Merrill
(1918) raised V. odorata and also noted its close similari-
ties to V. philippinensis, and he recognized V. littoralis and
V. odorata
as conspecific (Merrill 1923). These Villaria
species do not appear morphologically distinct and are
probably conspecific. Recently, Mulyaningsih and Ridsdale
(2004) described a diverging species of Hypobathrum
Blume as Villar ia, V. glomerata, which has five stigmatic
lobules and dioecious sexuality, in contrast to the common
bilobed stigma and hermaphrodite features of the genus. A
taxonomic revision of Villaria, presented below is thus
needed to understand this genus and provide a basis on
which to evaluate its relationships. This work is part of an
ongoing Philippine Rubiaceae project (Alejandro and
Liede 2003; Alejandro et al. 2008) particularly focusing on
endemic genera (Alejandro et al. 2010).
Materials and methods
Taxon sampling
This study was based on field observations and examina-
tion of more than 300 herbarium specimens of Villaria
(including type and recent collections) from the following
herbaria: A, CAHUP, G, GH, K, L, NY, P, PNH, PR and
US (abbreviations after Holmgren et al. 1990). Details of
the herbarium material consulted are listed in the taxo-
nomic treatment of each Vil laria species. Fieldwork was
conducted mostly in the type localities of Villaria found in
Luzon Island (Aurora, Isabela, Camarines Norte and
Quezon provinces). Twenty-seven collected Villaria
2 G. J. D. Alejandro et al.
123
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samples (herbarium specimens and material preserved in 70%
ethanol) were provided with a voucher (nos. 25051–25077) to
the University of Santo Tomas Herbarium (USTH). Leaf
material of five Villaria individuals (two isolates of V. glom-
erata and one isolate each of V. odorata, V. philippinensis and
V. rolfei) was dried in silica-gel for DNA extraction (Chase
and Hills 1991). We were able to obtain the sequences of four
of the six currently recognized Villaria species.
To reveal the tribal position of Villaria, we included also
sequences of related taxa in the phylogenetic analyses fol-
lowing the Ixoridinae II classification as outlined by Rob-
brecht and Manen (2006). Ixoridinae II sensu Robbrecht and
Manen outsizes the core Ixoridinae and consists of two large
sister groups, the Gardenieae alliance (Coffeeae L.,
Gardenieae DC., Octotropideae and Pavetteae A. Rich ex
Dumort) and the Vanguerieae alliance (Alberteae Hook.f.,
Ixoreae A. Gray and Vanguerieae Dumort). The genus
Luculia Sweet, which has been shown to be sister to all other
Rubiaceae (Bremer et al. 1999), was used as outgroup.
DNA extraction, PCR amplification and sequencing
Total DNA was extracted from silica gel-dried leaf tis-
sues using a DNeasy Plant Mini kit (Qiagen, Germany).
Extracted DNA was cleaned with the QiaQuick PCR
purification kit (Qiagen). Two different chloroplast
regions (rps16 and trnT-F) for which sequences were
available for representatives of Gardenieae and Van-
guerieae alliances were selected for amplification. The
primer pair used for amplification and sequen cing of the
rps16 intron was rps16-1F/rps16-2R (Oxelman et al.
1997). The trnT-trn-F region was amplified and
sequenced in two parts, first using the primer pair a/b
and the second using the primer pair c/f (Taberlet et al.
1991; Bremer et al. 2002).
PCR reactions were performed in volumes of 25 ll and
mixed as follows: 16.85 ll of dH
2
O, 2.5 ll of 109 PCR
buffer, 1.0 ll of MgCl
2
, 2.0 ll of dNTP, 1.0 ll of 10 lM
forward primer, 1.0 ll of 10 lM reverse primer and
0.15 ll of Taq DNA polymerase. To 24.5 ll of the mixture
was added 0.5 ll DNA template. PCR amplifications,
performed on a Biometra T-personal cycler (Germany),
started with an initial melting phase of 3 min at 94"C,
followed by 30–35 cycles of 1 min at 93"C, 1 min at 59"C
and 2 min at 72"C, and ended with a final extension phase
of 5 min at 72"C. In all PCR runs, one sample was run with
water instead of DNA as a negative control to test for
contamination. All sequences were retrieved by the com-
mercial services of Entelechon in Regensburg, Germany
(www.entelechon.com). EMBL accession numbers of all
new rps16 and trnT-F sequences of Villaria and sequences
taken from GenBank are listed in Table 1.
Table 1 Nucleotide sequence database accession numbers of taxa used in the phylogenetic analyses
Taxon GenBank accession number
rps16 trnT-F
Aidia micrantha (K. Schum.) Bullock ex F. White AF200974 AF201028
a
Alberta minor Baill. EF205637 EU817452
Aleisanthia rupestris (Ridl.) Ridl. AF242902 AF152660
Aleisanthiopsis distantiflora (Merr.) Tange EU817434 EU817453
Argocoffeopsis scandens (K. Schum.) Lebrun – DQ180568
b
Belonophora coriacea Hoyle – DQ180570
b
Bertiera guianensis Aubl. AF200983 AF152670
b
Canephora sp. – DQ180579
b
Coffea arabica L. AF004038 DQ153845
b
Coptosperma nigrescens Hook.f. AM117294 –
Coptosperma sp. – AM117355
Cremaspora triflora (Thonn.) K. Schum. AF200990 FM177173
b
Cyclophyllum deplanchei Hook.f. EF205640 EF205631
Diplospora polysperma Valeton AM117301 EU145538
Discospermum abnorme (Korth.) S. J. Ali & Robbr. – DQ180581
b
Feretia aeruginescens Stapf EU821618 EU145539
Fernelia buxifolia Lam. AF244892 EU145540
Gardenia hansemannii K. Schum. FM204720 –
Gardenia volkensii subsp. spathulifolia (Stapf & Hutch.) Verdc. – AM117360
Greenea corymbosa (Jack) Voigt AF242961 AF152657
b
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 3
123
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Data analysis
The rps16 and trnT-F sequences of Villar ia were assem-
bled and edited using CodonCode Aligner version 3.0.1.
All sequences were aligned manually with Se-Al v.1.0al
(Rambaut 1996). Ambiguous parts of the alignments were
excluded from datasets used for the analyses. Insertion–
deletion events inferred by eye were coded and used in
every analysis as a complementary partition following
Mouly et al. (2009). The aligned rps16 and trnT-F matrices
are available from the first author upon reque st.
Bayesian analysis was performed in MrBayes 3.1.2p
(Huelsenbeck and Ronquist 2001; Ronquist and
Huelsenbeck 2003; Altekar et al. 2004). For each DNA
region, the best-performing evolutionary models were
identified under three different model selection criteria:
Akaike information criterion (AIC) (Akaike 1974), AICc (a
second order AIC, necessary for small samples) and the
Bayesian information criterion (BIC) (Schwartz 1978). The
selected mode l for both rps16 and trnT-F partitions was the
general time reversible (GTR) model (Yang 1994), with
among-site substitution rate heterogeneity described by a
gamma distribution and all sites constraint to be variable.
These calculations were performed with MrAIC version
1.4.3 (Nylander 2004). For single-marker analysis, the
best-performing model was selected and five million
Table 1 continued
Taxon GenBank accession number
rps16 trnT-F
Greeniopsis discolor Merr. GQ981426 GQ981417
Hypobathrum racemosum (Roxb.) Kurz AM117318 FM207127
Ixora coccinea L. EF205641 EU817464
Ixora parviflora Lam. EU817449 EU817473
Kraussia floribunda Harv. AM117325 AM117368
Leptactina leopoldi-secundi Bu
¨
ttner EU821621 AY555088
a
Luculia gratissima (Wall.) Sweet EU817448 EU817472
Paragenipa lancifolia (Bojer ex. Baker) Tirveng. & Robbr. AF004066 AF152672
b
Pavetta barbertonensis Bremek. AF004069 AF152668
Peponidium cystiporon (Cavaco) Razafim., Lantz & B. Bremer EU817435 FN386322
Polysphaeria macrophylla K. Schum. – FM177172
Pouchetia baumanniana Bu
¨
ttner EU821625 EU145541
Pouchetia gilletii De Wild. AM117336 –
Psilanthus mannii Hook.f. – DQ180587
b
Rosenbergiodendron densiflorum (K.Schum.) Fagerl. AF201014 AF201061
a
Sericanthe sp. – DQ180593
b
Tarenna borbonica (Hend. & Andr.Hend.) Verdc. AF201024 AF152667
b
Tarennoidea wallichii (Hook.f.) Tirveng. & Sastre AF201025 AF201070
a
Tricalysia cryptocalyx Baker AF004088 AF152669
b
Vangueria madagascariensis J.F.Gmel. EU821636 AM117386
Villaria glomerata (Bartl. ex DC.) Mulyan. & Ridsdale (1)
c
JF827088 JF827093
Villaria glomerata (Bartl. ex DC.) Mulyan. & Ridsdale (2)
d
JF827089 JF827094
Villaria odorata (Blanco) Merr.
e
JF827090 JF827095
Villaria rolfei S. Vidal
f
JF827091 JF827096
Villaria philippinensis Rolfe
g
JF827092 JF827097
Xantonnea parvifolia (Kuntze) Craib – DQ180599
b
Information on origin and vouchers of most sequences has been published elsewhere, except for new sequences of Villaria species which is given
as footnotes
a
trnL-F intergenic spacer available
b
trnL gene and trnL-F intergenic spacer available
c,d
Luzon, Pangasinan prov., Bolinao Adube & Alejandro 25076 (USTH), 25077 (USTH)
e
Luzon, Quezon prov., General Nakar, de Leon & Alejandro 25055 (USTH)
f
Luzon, Isabela prov., Palanan, de Leon & Alejansro 25061 (USTH)
g
Luzon, Camarines Norte, Paracale, de Leon 25057 (USTH)
4 G. J. D. Alejandro et al.
123
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generations were run, with a sample frequency of 1,000
and four parallel chains. For combined rps16-trnT-F
analysis, model selection and settings were selected in the
same way as for single marker analysis (see above), but in
order to ensure that the analysis represented an adequate
sample of the posterior distribution, 30 million generations
were run. Partitions were unlinked so that each partition
was allowed to have its own set of parameters. The
Bayesian majority rules consensus tree calculat ion fol-
lowed after burn-in of 3,000 recorded generations. Groups
characterized by a posterior probability (PP) of more than
0.95 were regarded as strongly supported.
To compare the topologies inferred from Bayesian
analysis, parsimony analysis was conducted using PAUP*
version 4.0b (Swofford 2000). The maximum parsimony
(MP) trees were found by heuristic search, tree-bisection-
reconnection (TBR) branch swapping, using 10,000 ran-
dom addition sequences, with the MULTREES option on.
The consistency index (Kluge and Farris 1969) and reten-
tion inde x (Farris 1989) were calculated to estimate
homoplasy. Support values were calculated with bootstrap
(BS) analysis using 10,000 replicates, the MULTREES
option off, TBR branch swapping, and five random addi-
tion sequences. Groups receiving a BS value greater than
90% were considered strongly supported.
Combinability of the rps16 and trnT-F datasets was
evaluated using the incongruence length difference test,
also called the partition homogeneity test (Farris et al.
1994), as implemented in PAUP* (Swofford 2000). The
heuristic search was set to 500 replicates with ten random
addition sequences and nearest neighbour interchange
branch swapping.
Taxonomic study
Examination of reproductive morphology was based
mostly on preserved collections made during this project
using a Leica dissecting microscope. For the few species
for which no alcohol-preserved material was available,
morphological examination was done on parts rehydrated
from herbarium specimens. All other plant measurements
(e.g. leaves, stipules and inflorescences ) were taken from
dried specimens. Pollen grains obtained from recent col-
lections were prepared by the acetolysis method. For
scanning, scanning electron microscopic (SEM) images
from a Philips XL-30 system at 15 kV (for seeds) and a
Zeiss 1,530 system at 3 kV (for pollen) were used. For
counting chromosomes, root tips of three different plants of
V. odorata, propagated in the greenhouse of the Depart-
ment of Plant Systematics, University of Bayreuth, were
investigated after pretreatment with 0.002 M hydroxy-
chinoline following Tjio and Levan ( 1950), and staining
with carmine after Snow (1963). To depict a detailed
overview of the distribution of all recognized Villaria
species, the localities of all specimens based on informa-
tion from herba rium labels were mapped using Online Map
Creation (OMC; www.aquarius.geomar.de). The coordi-
nates were collected from gazetteers (www.nima.mil), and
were also checked in atlases.
In the genus description, character states in boldface are
considered implicit features and not repeated in the indi-
vidual species descriptions. The taxonomic key presented
for Villaria emphasized the most noticeable characters of
each species. Local names gathered through interviews and
labels from herbarium specimens are given for each species
with the standard abbreviation of Philippine local dialects
by Madulid (2001) in parentheses.
Results
Sequence variation of the two plastid regions
The rps16 and trnT-F analyses included 36 and 43
sequences, respectively. Five Villaria sequences for each
marker are newly published here. The sequences of rps16
intron of the sampled Villaria varied from 773 base pairs
(bp) (V. philippinensis
) to 808 bp (V. glomerata 2) while
the trnT-F region ranged from 1,594 bp (V. philippinensis)
to 1,667 bp (V. glomerata 1). Four gap-coded indels were
added to the datasets, one from the rps16 intron and three
from the trnT-F region (Table 2).
Genetic variation across the two cpDNA regions is low
allowing alignment without difficulty. Of the two chloropl ast
markers used, the trnT-F provided slightly more informative
sites consistent with earlier Rubiaceae reports (e.g. Razafi-
mandimbison and Bremer 2002; Alejandro et al. 2005;
Mouly et al. 2009). The aligned rps16 and trnT-F matrices
contained a total of 962 and 2,012 bp, respectively. The
rps16 dataset had 71 (7.38%) parsimony-informative sites
while the trnT-F dataset included a total of 141 (7.01%)
parsimony-informative characters (Table 2).
Phylogenetic analysis
Comparing the tree topologies of the separate rps16 and
trnT-F analyses (trees not shown here but available from
the first author), the rps16 tree was more congruent with
the trees generated from both MP and Bayesian inference
of the combined rps16-trnT-F dataset. In contrast to the
rps16 tree (not shown) and the combined tree (Fig. 1),
Razafimandimbisonia minor was placed sister to Octo-
tropideae and the sampled Gardenieae were unresolved in
the trnT-F tree (not shown). Nevertheless, the incongru-
ence length difference test indicated that the rps16 and
trnT-F datasets were significantly congruent (P = 0.256).
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 5
123
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Thus, we chose to analyse the consensus tree from the
combined dataset which yielded better resolution.
The aligned combine d matrix of 44 taxa had a total
length of 2,978 bp, of which 208 characters (7%) were
parsimony-informative, plus four gap-coded indels
(Table 2). The heurist ic search excluding uninformative
characters resulted in 217,500 most parsimonious trees,
each with a length of 375 step s (Table 2). The consistency
index and retention index were 0.70 and 0.85, respectively.
The topologies of the MP strict consensus and the Bayesian
majority rule consensus tree were congruent, except for
three nodes indicated by asterisks within the Coffeeae,
Pavetteae and Octotropideae clades (Fig. 1) that were left
unresolved by the MP analysis.
In general, the monophyly of the included tribes is
robustly supported (Fig. 1), consistent with previous stud-
ies in Ixoroideae (Andreasen and Bremer 2000; Robbrecht
and Manen 2006; Bremer 2009). Similar to earlier studies,
the relationships between Coffeeae, Gardenieae-Pavetteae
and Octotropideae are left unresolved within the Garden-
ieae alliance (Fig. 1). To avoid misleading information,
Gardenieae forms here a clade due to a restricted sampling,
whilst numerous studies retrieved a nonmonophyletic
Gardenieae group using broader generic samplings (e.g.
Persson 2000, Robbrecht and Manen 2006; Bremer 2009;
Bremer and Eriksson 2009).
In the separate plastid analyses (trees not shown) and
combined tree (Fig. 1), all sampled Villaria species are
resolved as monophyletic with strong support (PP = 1.00,
BS = 99%) and nested within Octotropideae (Fig. 1 ),
consistent with Robbrecht et al. (1993) based on mor-
phology. Villaria is placed in a group containing Hypo-
bathrum and Pouchetia with strong support in Bayesian
analysis (PP = 0.96), inconsistent with the informal group
placement of the genus by Robbrecht and Puff (1986). This
group forms a sister relationship (strongly supported in
Bayesian analysis, PP = 0.97) with a poorly suppor ted and
unresolved group (PP = 0.54) containing Feretia Delile,
Fernelia, Kraussia Harv. and Polysphaeria. A strongly
supported sister taxa relationship (PP = 1.00, BS = 94%)
between Canephora Juss. and Paragenipa was found basal
together with Cremaspora (Fig. 1). Following the recom-
mendation of Robbrecht and Manen (2006) on the place-
ment of Cremaspora in Octotropideae, the tribe is strongl y
supported (PP = 1.00, BS = 90%) as monophyletic.
Morphology of Villaria
Plants of Villaria are usually shrubs (Fig. 2a) to small-
sized trees that are vegetatively almost fully glabrous.
Young stems are flattened with paired lateral branches that
often originate in a supraaxillary position (Fig. 2b). Lateral
branches are subterete on the basal node and flattened on
the apical node. Leaf blades vary from elliptic to ovate, or
lanceolate, and are mostly coriaceous. Petioles are not
articulate and do not exceed 1 cm in length. Interpetiolar
stipules are entire, are usually triangular, are basally fused
around the node, rarely exceed 1 cm in length, and are
prominently keeled (Fig. 2b). Several colleters at the inner
bases of the stipules show a linear-subulate to lanceolate
shape, and their secretion turns brownish with age.
The usually paired inflorescences are not always axillary,
occasionally occurring slightly above the node (supraaxil-
lary), similar to other reported Octotropideae (e.g. Robbrecht
et al. 1993; Tosh et al. 2008). Inflorescences are variable and
constitute either simple or compound dichasial cymes (V.
odorata, Figs. 2c, 3a), glomerules (V. glomerata), or are,
rarely, uniflorous (V. acutifolia). They are usually peduncu-
late, rarely subsessile (V. glomerata). Although the flowers of
Table 2 Matrix characteristics and tree statistics of separate and combined datasets
rps16 trnT-F Combined data
No. of taxa 36 43 44
Matrix length 962 ? 1 gap-coded indel 2,012 ? 3 gap-coded indels 2,974 ? 4 gap-coded indels
No. of included characters 818 ? 1 gap-coded indel 1,741 ? 3 gap-coded indels 2,559 ? 4 gap-coded indels
No. of parsimony-informative characters 70 ? 1 gap-coded indel 138 ? 3 gap-coded indels
(trnT-L = 62; trn L = 27; trnL-F = 52)
208 ? 4 gap-coded indels
No. of most parsimonious trees 783 216,500 217,500
Tree length 134 237 375
Consistency index 0.68 0.71 0.70
Retention index 0.83 0.87 0.85
Fig. 1 Majority rule consensus tree inferred from Bayesian analysis
of the combined rps16-trnT-F dataset. These results are congruent
with parsimony analysis, except for the three nodes within COF, OCT
and PAV clades marked by asterisks. Numbers above branches
indicate Bayesian posterior probabilities and those below the
branches parsimony bootstrap support values. Current tribal place-
ments of included taxa according to Robbrecht and Manen (2006) as
follows: ALB Alberteae, COF Coffeeae, GAR Gardenieae, OCT
Octotropideae, PAV Pavetteae, VAN Vanguerieae. Tribes abbreviated
as ALE Aleisanthieae, GRE Greeneeae and IXO Ixoreae follow Mouly
et al. (2009) and Alejandro et al. (2010). The Villaria clade is
indicated in grey
c
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V. glomerata have been described as unisexual (Merrill 1918;
Mulyaningsih and Ridsdale 2004), all Villaria species have
hermaphroditic flowers. Flowers are five-, rarely six-merous,
odourless, always bibracteate, and corolla aestivation is con-
torted to the left (Fig. 3b). The calyx consists of a short tube
bearing five lobes. Calyx tubes do not exceed 5 mm in length,
and are extremely short (1 mm or less) in V. glomerata. While
the outer parts of calyx tubes are mostly glabrous, except in V.
acutifolia and V. glomerata which are puberulous, the inner
side is usually densely pilose extending up to the base of the
calyx lobes. The calyx lobes are usually short, depressed ovate
(Fig. 3b), rarely long, oblanceolate (V. acutifolia), and
sometimes with basal colleters within. The corolla is thick and
leathery, funnel-shaped, rarely cylindrical (V. acutifolia),
white turning ivory with age, always glabrous on the outer
part, and densely pilose over the upper half of the corolla tube
apart from a glabrous lower area inside. The corolla lobes are
mostly ovate to rounded, not exceeding 1 cm in length and
width, and always glabrous on both sides, except for the
basally densely pilose adaxial side. The anthers are five, each
with two thecae, epipetalous (Fig. 3d), partially exserted
(Figs. 2c, 3d), dorsimedifixed, glabrous and sessile. The style
is short, cylindrical and glabrous (Fig. 3e, f). The stigmatic
lobes are elliptic to oblong, partially exserted, always densely
pilose abaxially (Figs. 2c, 3e, f), but adaxially glabrous, or
rarely papillate (V. leytensis). As in some Octotropideae, the
stigma lobes are entirely adnate before maturity and partly
separate on the apical portion only in mature styles (Figs. 2c,
3e, f). The morphology of styles and anthers suggests sec-
ondary pollen presentation (Puff et al. 1996).
Microscopic sectioning made on the ovaries of Villaria
showed a unilocular condition with two parietal placentas
(Fig. 3g). The placentas are irregularly much branched and
contain many ovules. The ovules are strictly horizontally
oriented (Fig. 3g), more or less immersed in the placenta,
with superior radicles. Fruits are berry-like with a leathery
mesocarp and membranous endocarp. The fruits are
ellipsoidal to globose, crowned by the persistent calyx,
bibracteate, green when young, brown when mature,
usually glabrous (Figs. 2d, 3h), rarely puberulous (V.
acutifolia, V. glomerata).
SEM investigations of Vil laria pollen showed three-
colporate, spheroidal to suboblate grains, measuring
between 20 and 28 lm in diameter, with a tectum that is
either microreticulate (Fig. 4a, c) or perforate (Fig. 4b, d),
consistent with reported pollen features of six Octotropi-
deae (Dessein et al. 2005). The small seeds of Villaria are
numerous, angular–ovoid, laterally flattened (Fig. 4e)
(golden) brown, and covered with fibrous testa (Fig. 4e, f)
enclosing an entire endosperm (Fig. 4g).
Chromosomes
Based on the analysis of mitotic metaphase plates, we
counted 2n = 44 chromosomes in three different vouchers
of V. odorata (Espino et al. 25051 et 25054, and de Leon
and Alejandro 2505; Fig. 4h). These counts represent the
first for the whole genus. The chromosomes are fairly
uniform in size, about 0.9–1.6 lm in length on average.
Discussion
Phylogenetic position of Villaria
Our phylogenetic results do not support a close relationship
between Villaria and Gardenia (Rolfe 1884) or the inclu-
sion of Villaria in the tribe Gardenieae as proposed by
Fig. 2 Villaria odorata. a Habit. b Stem showing stipule at the node with paired axillary petioles, paired supraaxillary lateral branches located
above the stipule. c Flowering branch with front view of flower. d Fruit. Photos taken by G.J.D. Alejandro (a, c, d), and U. Meve (b)
8 G. J. D. Alejandro et al.
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Schumann (1891), Verdcourt (1958) and Bremekamp
(1966). This study is the first to support the inclusion of
Villaria in Octotropideae using molecular data, consistent
with Robbrecht (1980) and Robbrecht and Puff (1986)
based on morphology. Villaria shares characters common
in Octotropideae such as paired, axillary or supraaxillary
inflorescences, hermaphrodite flowers, left-contorted aes-
tivation, secondary pollen presentation, numerous ovules,
fleshy fruits, superior embryo radicles, entire endosperm,
fibrous exotesta, and three-colporate pollen (cf. Robbrecht
Fig. 3 Villaria odorata (Blanco) Merr. A flowering branch; B corolla
bud showing aestivation; C flower; D, E opened corolla; F style;
G ovary in longitudinal section showing ovule orientation; H whole
fruit; I fruit in longitudinal section; J fruit in cross-section. From E.P.
de Leon & G.J.D. Alejandro 25059 (PNH), except G from E.P. de
Leon & G.J.D. Alejandro 25065 (USTH). All drawn by N. Diego,
except B, J and G by U. Meve
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 9
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1988; Bridson and Verdcourt 2003; Ruhsam and Davis
2007).
However, our molecular results (Fig. 1) do not support
the placement of Villaria among the ‘‘primitive’’ genera
(together with other Asian representatives plus Ramosma-
nia) of Octotropideae recognized by Robbrecht and Puff
(1986). In contrast to the informal groups, our com bined
tree (Fig. 1) implies a close association betwee n Villaria,
Hypobathrum and Pouchetia, a relationship that receives
considerable support in the Bayesian analysis (PP = 0.96).
The sister taxa relationship between the African Pouchetia
and Asian Hypobathrum was earlier demonstrated by An-
dreasen and Bremer (2000) and Bremer and Eriksson
(2009). Morphologically, these three genera share her-
maphrodite flowers and fibrous exotesta cells. Both Hyp-
obathrum and Pouchetia were placed in the ‘‘central’’
group (Robbrecht and Puff 1986) distinguished by smaller
fruits, few ovules, and thickening of the radial walls of
the exotesta cells. Also, the weakly suppor ted group
(Feretia to Polysphaeria, Fig. 1) includes representatives
of the ‘‘primitive’’ (Fernelia), ‘‘central’’ (Feretia), and
‘‘advanced’’ (Kraussia and Polysphaeria) groups. These
four genera share the absence of fibrous exotesta cells.
Obviously, our combined tree (Fig. 1) does not conform to
this informal grouping of the tribe. With the exception of
Canephora, Octotropideae placed outside the Hypoba-
thrum-Pouchetia-Villaria group are characterized by the
absence of a fibrous exotesta. Group recognition in the tribe
is possibly more reasonable based on the structure of
exotesta. Octotropideae is a large tribe and comprehensive
studies of many genera, particularly Asian representatives,
are still lacking. Further investigation is clearly needed to
pinpoint taxonomically significant characters in the tribe
for a better understanding of their relationships.
In addition, our combined tree (Fig. 1) strongly supports
Canephora and Paragenipa as sister taxa (PP = 1.00,
BS = 96%) that form an unresolved clade together with
Cremaspora
and the rest of Octotropideae. A close rela-
tionship between Cremaspora and Paragenipa was also
earlier depicted by Andreasen and Bremer (1996) and
Persson (2000). Our results further confirm the close rela-
tionship between Cremaspora and Octotropideae,
Fig. 4 SEM images of Villaria
seed and pollen morphology,
and light microscopic image of
Villaria odorata chromosomes.
a, b Three-colporate pollen
showing microreticulate
ornamentation (detail in b top
right)(V. odorata). c, d Pollen
showing elliptic pore and
perforate ornamentation (detail
in d top left)(V. glomerata). e–
g Who le seed (e), detail of
fibrous exotesta (f), and section
of seed (g)(V. odorata).
h Metaphase of a somatic cell of
the root tip meristem showing
2n = 44 chromosomes (V.
odorata). From E.P. de Leon &
G.J.D. Alejandro 25061
(USTH) (a , b), K. Adube &
G.J.D. Alejandro 25076
(USTH) (c, d), E.P. de Leon &
G.J.D. Alejandro 25065 (L,
PNH) (e–g), and Espino et al.
25054 (h)
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consistent with several other studies (Andreasen and
Bremer 2000; Persson 2000; Robbrecht and Manen 2006;
Davis et al. 2007; Tosh et al. 2008; Bremer and Eriksson
2009).
Salient features of Villaria
Villaria forms a strongly supported (PP = 1.00,
BS = 99%) monophyletic group within Octotropideae. As
far as we know, although comprehensive data on ovary
morphology for the tribe are lacking, Villaria is the only
representative characterize d by unilocular ovaries with
biparietal placentation, a rare feature not only in the tribe
but in Rubiaceae as a whole. Unilocular ovaries with
biparietal placentation are also present in some species of
Randia (e.g. Gustafsson and Persson 2002). Robbrecht and
Puff (1986) reported that Poucheti a and Fernelia occa-
sionally possess parietal placentation, but both genera and
all other known members of the tribe have bilocular ovaries
(e.g. Canephora, Feretia, Hypobathrum, Kraussia, Par-
agenipa, Polysphaeria). Recently, the ovaries of Flageni-
um (Ruhsam and Davis 2007) and Petitiocodon (Tosh et al.
2008) were demonstrated to be bilocular. In Rubiaceae,
very few genera (e.g. Theligonum L.) are reported to pos-
sess a unilocular ovary (Robbrecht 1988). This character
seems to have evolved independently within the family.
Octotropideae were previously noted to have relatively
uniform ovaries with pendulous ovules attached to an
apical placenta (Robbrecht and Puff 1986). This is true for
Madagascan (Canephora, Chapelieria A. Rich ex DC.,
Gallienia, Jovetia and Lemyrea), African (Feretia, Galin-
iera Delile, Kraussia, Lamprothamnus Hiern, Polysphaeria
and Pouchetia), Seychellean (
Paragenipa), Mascarene
(Fernelia), and two Asian (Nargedia and Octotropis)
Octotropideae. On the contrary, Villaria species are char-
acterized strictly by numerous horizontally arranged ovules
developing from richly branching parietal placentas
(Figs. 3g, j). Such an ovule arrangement was also noted in
Ramosmania (Tirvengadum 1982), although Ruh sam and
Davis (2007) recently described the lower ovules in each
locule as slightly pendulous. Noticeable variation in ovary
morphology is also described in Flagenium possessing
either two rows of ovules (the upper erect and lower pen-
dulous) or a set of horizontal ovules (Ruhsam and Davis
2007), whereas Petitiocodon is distinct with two lateral
ovules attached to an elongated placenta (Tosh et al. 2008).
Most likely, divergence of the ovary may be present in
other poorly know n genera of the tribe, especially the
Asian members.
Furthermore, Villaria is one of the few genera in
Octotropideae characterize d by numerous ovules, indu-
mented stigmas, unribbed fruits, and leathery texture of the
fruit walls. The short bifid stigma at maturity, superior
radicle orientation, pollen morphology and seed morphol-
ogy in Villaria are reported here for the first time. However,
these characters occur in other genera of the tribe as well.
Based on our observations, the salient features of Vil-
laria include: vegetatively almost glabrous; young stems
flattened; lateral branches supraaxillary; petioles not
articulate; inflorescences paired, axillary or supraaxillary,
simple or compound cymes, sometimes uniflorous or
glomerulous; flowers hermaphroditic, five-merous, sub-
tended by pairs of bracts; corollas left-contorted in bud,
usually funnel-shaped; anthers partially exserted; stigmas
partially exserted, abaxial ly densely pilose, partially sepa-
rating at maturity; ovaries unilocular, with parietal pla-
centation; ovules numerous, horizontally arranged; fruits
with persistent calyces, leathery mesocarp, membranous
endocarp; seeds with fibrous exotesta, entire endosperm,
superior radicle.
The Villaria species
In this revision, we recognize a total of five Villaria spe-
cies: V. acutifolia, V. fasciculiflora, V. glomerata, V. leyt-
ensis and V. odorata. Within the genus, the morphology of
calyx, corolla, inflorescences, and less the leaves, are
useful to determine the species. Almost all Villaria species
are vegetat ively glabrous, except for a few scattered tric-
homes in the young branches of V. acutifolia and V.
glomerata, the leaf margins of V. leytensis, and pilose
stipules in V. glomerata. Leaf blades are usually elliptic to
ovate, rarely lanceolate to narrowly elliptic (V. glomerata).
Small leaf blades (length not exceeding 15 cm, width 5 cm
or less) are generally exhibited by V. acutifolia,
V. leytensis
and V. glomerata, while larger ones (length up to 25 cm,
width up to 9 cm) are found in V. fasciculiflora and V.
odorata. The stipules are invariably triangular to ovate and
their average size is 1 cm.
The paired inflorescences of Villaria are not always
axillary, but are mostly placed 1–4 mm above the node,
and are thus supraaxillary, which is also observed in sev-
eral Octoropideae (e.g. Tosh et al. 2008). The inflorescence
type (as mentioned in the Results) is also significant for
species recognition in Villaria. The shape of calyx lobes is
consistently depressed ovate, rarely oval in four Villaria
species (V. fasciculiflora, V. odorata, V. leytensis and V.
glomerata) not exceeding 3 mm in length. Prominently
long calyx lobes (up to 15 mm) exceeding the corolla
length, and with a distinct midnerve, are present in V.
acutifolia. Villaria acutifolia also differ in the shape of
corolla lobes which are elliptic to ovate against the usual
rounded corolla lobes in the other Villaria species.
Of particular interest is the sexuality of V. glomerata.
Previous authors describing the species under different
names (e.g. Blanco 1837; Schumann 1891) simply
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 11
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described the stamen and pistil but did not comment on the
sexuality. Merrill (1918) described the dioecious nature of
the species, with numerous, short pedicellate male flowers,
and a few, long pedicellate female flowers. Mulyaningsih
and Ridsdale (2004), provided with only limit ed herbarium
specimens and without seeing the type specimen, simply
followed Merrill’s observations and added that the female
flowers possessed five stigma lobules. Most of the speci-
mens examined by Mulyaningsih and Ridsda le (2004) were
collections from Bataan, Luzon. Recent field collection on
the site showed a common existence of one Lasianthus
species which vegetatively resembles V. glomerata and
matches for the presence of five stigma lobes described in
the ‘‘female flower’’ of the species. Additionally, exami-
nation of several designated male flowers of V. glomerata
showed the presence of both anthers and style. Our findings
confirm two stigma lobes and hermaphrodite sexuality in V.
glomerata. The divergent forms appear in the flowering
and fruiting stages. The pedicels develop very late (as
observed in the long pedicellate ‘‘female flower’’) so that
young inflorescences are glomerulous and subsessile or
short pedicellate. Corollas are shed off after anth esis
Fig. 5 Distribution of Villaria
acutifolia, V. fasciculiflora, V.
glomerata, V. leytensis and V.
odorata
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allowing the development of pedicels. A similar situation
was observed in the monotypic Octotropis (Robbrecht et al.
1993). Accordingly, the description of V. glomerata is
amended below.
Based on comparative morphological assessment of the
three Villaria species in the field and b y recently collected
herbarium vouchers and alcohol-preserved material, we
here consider V. p hilippinensis and V. rolfei as conspecific
with, and as synonyms of, V. odorata based on morphol-
ogy. These three taxa are indistinguishable in terms of
vegetative and reproductive morphology. Minor variation
exists only in the shape of the leaves, and this is the reason
earlier authors separated these species.
Chromosome data for Octotropideae are restricted to
some species of the genera Ramosmania (Owens et al.
1993), Galiniera, Feretia, Kraussia and Polysphaeria
(Kiehn 1985). All these samples have been shown to pos-
sess a diploid chromosome number of 2n = 22. The only
exception is n = 24 (x = 12) reported for Hyptianthera
stricta (Sarkar et al. 1982), but this unexpected count needs
to be corroborated.
The basic chromosome number of x = 11 is the most
widespread in flowering plants, the Gentianales, and is
also consistent in several tribes of Rubiaceae (e.g. De
Block 1998; Kiehn 1985; Philip and Mathew 1988;
Robbrecht 1988). Villar ia odorata also has x = 11, but
is a tetraploid with 2n = 44. This condition is fairly
widespread in Rubiaceae–Ixoroideae, especially in Van-
guerieae where nearly all investigated taxa are tetraploid
(cf. Kiehn 1985, 1996), but is assured for the first time
in Octotropideae. However, whether the whole genus is
tetraploid or just V. odorata is still to be tested with
additional representatives of Villaria, the latter being a
species that also typically behaves like a (neo)pol yploid
(cf. Levin 1983) with its highest abundance by far and
widest distribution within the genus from Central Luzon
and Calabarzon region extending northwards to Cagayan
Valley up to the Babuyan and Batanes group of islands ,
and southwards to Samar (Visayas) and Surigao (Mindanao)
(Fig. 5
). The other four Villaria species are generally
restricted to smaller areas of mostly single islands, such as
V. acutifolia in Davao (Mindanao), V. fasciculiflora in
Isabela (Luzon), V. glomerata mostly in Central Luzon, and
V. leytensis in Leyte (Visayas).
The distribution of V. acutifolia and V. leytensis, known
only from their type localities (Fig . 5), even gives the
impression of being relictual. Unfortunately, the herbarium
specimens reveal little of the habi tat of the plants. Recent
collections are predominantly found in coastal forests
(primary or secondary forests behind the beach ), gully
forests, forest margins, scrub on upper beaches (above the
high-tide mark), typically on clayey or sandy soils. Apart
from ecology, fruit morphology suggests dispersal by
water. The solid green fruits of Villaria are possibly spread
by (sea)water. The leathery pericarp of Villaria fruits,
turning brown at maturity, and the membranous-porose
endocarp seem to point to hydrochory, but not necessarily
to endochory. Also, seedlings have been tested to be very
tolerant of NaCl (Meve, personal observation). Villaria,
however, does not thrive in mangrove vegetation.
The taxonomic revision of Villaria is presented below.
Taxonomic treatment
Villaria Rolfe, J. Linn. Soc., Bot. 21: 311. 1884. TYPE
species: V. philippinensis Rolfe.
Shrubs to small-sized trees; young stems flattened;
paired lateral branches often supraaxillary, basally subter-
ete, apically flattened, green when young (dark-)brown
when mature, usually glabrous , rarely puberulous. Leaf
blades elliptic to ovate, or lanceolate (sub)coriaceous,
glabrous on both sides, rarely puberulous on midrib and
veins underneath; petioles short, glabrous. Stipules basally
fused, interpetiolar, triangular to ovate, prominently
keeled, glabrous on both sides, green, rarely indumented,
apically sparsely ciliate; colleters linear to lanceolate,
adaxially densely at the base.
Inflorescences axillary or supraaxillary, simple or
compound dichasial cyme, rarely uniflorous, or glome-
rulous, pedunculate, rarely subsessile. Flowers hermaph-
rodite, five-merous, rarely six-merous, odourl ess; pedicels
short, flat, bibracteate. Hypanthium short, obconical to
urceolate. Calyx tube glabrous or puberulous outs ide,
usually densely pilose inside extending to the base of the
calyx lobes; lobes usually depressed ovate, rarely
oblanceolate to narrowly elliptic, margins ciliate. Corolla
left contorted in bud, white; tube infundibular, rarely
cylindrical, glabrous outside, densely pilose on the upper
part inside; lobes mostly ovate to rounded, margins
sparsely ciliate. Anthers 5, epipetalous, partially exserted,
dorsimedifixed, linear, apex obtuse, base sagittate, gla-
brous, sessile. Pollen three-colporate, spheroidal to sub-
oblate, tectum microreticulate or perforate. Ovary
unilocular, with biparietal, irregularly much branched
placentas; ovules numerous, horizontally oriented; radicle
superior (micropyle upward); style glabrous; stigma
elliptic to oblong, entirely adnate, partly splitting when
mature, partially exserted, always densely pilose outside,
glabrous, or rarely papillate inside. Fruits fleshy, berry-
like, ellipsoid to globose, crowned with persistent calyx,
green, brown when ripe, usually glabrous, rarely pube-
rulous, mesocarp leathery, endocarp membranous, stalk
bibracteate. Seeds angular-ovoid, laterally flattened, small
(golden) brown, with fibrous exotesta.
Chromosome number: 2n = 44 (for V. odorata).
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 13
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Key to the Villaria species
1a. Calyx lobes oblanceolate to narrowly elliptic, mid-
nerve prominent, 8–15 mm long, longer than corolla;
corolla tube cylindrical; corolla lobes elliptic to ovate.
1. V. acutifolia.
1b. Calyx lobes depressed ovate to oval, midnerve
absent, 1.5–3 mm long, shorter than corolla; corolla
tube funnel-shaped; corolla lobes rounded.
2a. Leaf blades lanceolate to narrowly elliptic;
stipules pilose on both sides; inflorescences
glomerulate; corolla lobes 1–2.5 mm long.
3. V. glomerata.
2b Leaf blades elliptic to ovate, or obovate; stipules
glabrous on both sides; inflorescences three-
flowered, simple to compound dichasial cymes,
or uniflorous; corolla lobes 3–7 mm long.
3a. Leaf apices narrowly to abruptly acumi-
nate; stipules 10–18 mm long; inflores-
cences subsessile, never uniflorous; calyx
tube 1–2 mm long.
2. V. fasciculiflora.
3b. Leaf apices broadly acute to acuminate;
stipules 5–10 mm long; inflorescences
long pedunculate, sometimes uniflorous;
calyx tube 3–5 mm long.
4a. Leaf blades 5–9.5 9 2.5–3.5 cm,
petioles 3–5 mm long; corolla tube
3–4 9 2.5–3 mm; corolla lobes
4–4.5 9 4 mm; stigma adaxially
papillate; fruits 9–11 9 8–9 mm
4. V. leytensis.
4b. Leaf blades 8–13(–20.5) 9 (3.5–)
6–9 cm, petioles 5–10 mm long; cor-
olla tube 5–6 9 5 mm; corolla lobes
5–7 9 5–6 mm; stigma adaxially gla-
brous; fruits 14–16 9 10–14 mm.
5. V. odorata.
1. Villaria acutifolia (Elmer) Merr., Philipp. J. Sci. 5:248.
1910; Leafl. Philipp. Bot. 3:1007. 1911; Enum. Philipp. Pl.
3:532. 1923. : Gardenia acutifolia Elmer, Leafl. Philipp.
Bot. 1:6. 1906. TYPE: Philippines, Mindanao, Dav ao, Pa-
dada [06"38
0
N 125"22
0
E], March 1904, Copeland E.B. 437
(holotype, PNH, destroyed; lectotype, designated here,
NY!; isolectotype, US!).
Small trees, up to 4 m high; branches glabrous to
puberulous. Leaf blades lanceolate, or elliptic to ovate (2–
)5–15 9 (2–)3–5 cm, subcoriaceous, glabrous on both
sides; apex acuminate; base rounded to acute; margins
glabrous; secondary veins 9–11 pairs; petioles 2.5–5 mm
long, glabrous. Stipules o blong to triangular, keel
prominent from the tip along one-quarter of length,
9–15 9 5–9 mm, glabrous on both sides; colleters
numerous, 0.6–1 mm long.
Inflorescences axillary, mostly uniflorous, rarely three-
flowered simple cymes. Flower pedicels 7–15 mm long,
puberulous; bracts lanceolate to elliptic, apex acuminate to
rounded, 2–4 9 1–2 mm, puberulous outside, glabrous
inside, margins sparsely ciliate. Calyx tube 4–5 9
2–2.5 mm, puberulous outs ide; lobes oblanceolate to nar-
rowly elliptic, midnerve prominent, 8–15 9 2–3.5 mm,
exceeding the corolla, glabrous on both sides, margins cili-
ate. Corolla tube cylindrical, 4–4.5 9 2–2.5 mm; lobes
elliptic to ovate, 4 9 2.5 mm, glabrous on both sides.
Anthers 2.5–3 9 1 mm. Style 1.5–2 mm long; stigma
oblong, 3.5–4 9 1.5 mm, glabrous inside. Fruits globose,
9–13 9 8–12 mm, glabrous or puberulous; peduncle
1–2 cm long, glabrous. Seeds ovat e to elongate,
2–3 9 1–1.2 mm.
Distribution and habitat: Mindanao (restricted in Davao,
Padada). Herbarium specimens have no information on the
habitat, but Padada is near the boarder of Davao Gulf.
Probably a coastal species, on clay-sandy soils (Fig. 5).
Local name: Tango (BisC.).
Phenology: Flowering March–June; fruiting May–
August.
Discussion: This species can be easily recognized by its
usually uniflorous inflorescences and long calyx lobes
exceeding the corolla length. Elmer (1906) noted that V.
acutifolia might be a form of V. odorata. However, the two
species are disparate vegetatively, and more so reproduc-
tively. Villaria acutifolia appears to be locally rare and
restricted to Padada, Davao of Mindanao. It is presently
known only from three historical specimens collected
between 1903 and 1905. As no specimen has been col-
lected for more than 100 years and in spite of two attempts
by G.J.D.A. in 2007 and 2010 to collect in the type locality
and nearby provinces of Padada, this species could be
extinct.
Additional specimen examined: Mindanao: Davao Prov.,
Padada, 06.38
0
N, 125"22
0
E, 28 Jun. 1905, Williams 2975 (A,
GH, K, NY \4 sheets[ , US); Davao Prov., without exact
locality, Apr. 1903, DeVore & Hoover 123 (US).
2. Villaria fasciculiflora Quisumb. & Merr., Philipp.
J. Sci. 37:207. 1928. TYPE: Philippines, Luzon, Isabela,
San Mariano, Dibuluan [16"58
0
N 122"00
0
E], March 25,
1926, Ramos M. and Edan
˜
o G. 46775 (holotype, PNH,
destroyed; lectotype, designated here, K!; isolectotypes,
NY!, US!).
Shrubs, about 2 m high; vegetative parts glabrous. Leaf
blades elliptic to ovate, 13–23.5 9 4–8 cm, coriaceous,
pale-brown above, reddish-brown beneath; apex narrowly
to abruptly acuminate; base acute to rounded; secondary
veins 10–13 pairs, darker than the blade; petioles 8–10 mm
14 G. J. D. Alejandro et al.
123
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long. Stipules triangular to widely ovate, keel prominent
from the tip along half of length, 10–18 9 5–9 mm; coll-
eters few to numerous, 1–1.5 mm long.
Inflorescences axillary, or supraaxillary 1–2 mm above
the nodes, three-flowered simple cymes; peduncle 1–3 mm
long, glabrous. Flower pedicels 2–12 mm long, glabrous;
bracts triangular to ovate, apex acuminate to rounded,
2–3.5 9 3 mm, glabrous outside, basally sparsely pilose
inside, margins ciliate. Calyx tube 1–2 9 3–4 mm, gla-
brous outside; lobes depressed ovate, 1.5–2 9 2.5–3 mm,
glabrous on both sides, margins sparsely ciliate. Corolla
tube shortly infundibular, 4–5 9 4–4.5 mm wide on the
upper part; lobes orbicul ar, 3–4.5 9 3–5 mm, glabrous on
both sides except the basally pilose inside. Anthers
4 9 1 mm. Style 3–3.5 mm long; stigma narrowly elliptic,
4–5 9 1–1.5 mm, glabrous inside. Fruits unknown.
Distribution and habitat: Luzon (restricted in Isabela
and Rizal provinces); in secondary forest, on forest mar-
gins, on clayey-sandy soils; 700 m asl (Fig. 5).
Local name: Otto (Ibg.).
Phenology: Flowering February–April; fruiting probably
June–August.
Additional specimen examined: Luzon: Rizal Prov., Mt.
Angilog, 14"40
0
N, 121"15
0
E, Apr. 1922, M. Ramos s.n.
(K).
Discussion: Villaria fasciculiflora is easily recognized
by its shortly peduncled cymes, subsessile flowers, narrow
to abrupt leaf apices, and large stipules. This species
appears to be restricted to Luzon and locally rare. It is only
known from the type collection (Isabela province, Ramos
and Edan
˜
o 46775) and one herbarium sheet from Kew
recently identified by us (Rizal province, Ramos s.n.), also
in flowering stage.
3. Villaria glomerata (Bartl. ex DC.) Mulyan. & Rids-
dale, Reinwardtia 12:195–197. Fig. 1. 2004. : Platyme-
rium glomeratum Bartl. ex DC., Prodr. 4:619.
1830. : Hypobathrum glomeratum (Bartl. ex DC.) K.
Schum., in H.G.A. Engler & K.A.E. Prantl., Nat. Pflan-
zenfam. 4(4):156. 1891. TYPE: Philippines, Luzon, without
exact locality and date of collection, Haenke s.n. (holotype,
PR! \ 3 sheets [).
Serissa myrtifolia Blanco, Fl. Filip. [F.M. Blanco]:164.
1837. TYPE: Philippines, Luzon, Bulacan, Baliuag, Jan.
1910, Merrill 71 (neotype, designated here, US).
Serissa pinnata Blanco, Fl. Filip. [F.M. Blanco]:163.
1837. : Gardenia pinnata (Blanco) Merr., Philipp. Gov.
Lab. Bur. Bull. 27:53. 1905. TYPE: Philippines, Luzon,
Bulacan, Angat, July 1914, Merrill (Species Blancoanae
223) (neotype, designated here , US!; isoneotypes, L!,
NY!).
Remijia obscura Blanco, Fl. Filip. [F.M. Blanco] 2:116 .
1845. : Randia obscura (Blanco) Fern.-Vill., Fl. Filip.
[F.M. Blanc o] 3, 4(13A):108. 1880. : Gardenia obscura
(Blanco) Vidal, Phan. Cuming. Philipp. 18:119. 1885.
TYPE: Philippines, Tarlac, Murcia, Vidal 387 (neotype,
designated here, A).
Remijia angatensis Blanco, Fl. Filip. [F.M. Blanco]
2:115. 1845. : Randia angatensis (Blanco) Fern.-Vill.,
Fl. Filip. [F.M. Blanco] 3, 4(13A):108 1880. TYPE: Phil-
ippines, Luzon, Bulacan, Angat, July 1914, Merrill (Spe-
cies Blancoanae 688) (neotype, designated here, US;
isoneotype, L).
Shrubs or small trees, 2–3 m high; branches glabrous or
puberulous. Leaf blades lanceolate to narrowly elliptic,
7–14 9 2–5 cm (sub)coriaceous, glabrous on bo th sides, or
sparsely pubescent on the midrib and veins beneath; apex
narrowly acuminate; base acute or cuneate; margins gla-
brous; secondary veins 9–12 pairs; petioles 2–5 mm long,
glabrous or puberulous. Stipules triangular–ovate, keel
prominent from the tip along one-half to one-third of the
length, 2–7 9 1.2–4 mm , abaxially sparsely pilose on the
keel, adaxially densely pilose at the base and keel; colleters
numerous, 0.4–0.6 mm long.
Inflorescences axillary, or supraaxillary 2 mm above the
nodes, many-flowered glomerule; peduncles subsessile in
flower, but up to 1–2.4 cm long in fruiting stage, puberu-
lous. Flower pedicels subsessile, puberulous; bracts trian-
gular to ovate, apex rounded, 1–2.2 9 0.5–1.5 mm, with
few colleters at the base, sparsely to densely pilose on both
sides, margins sparsely ciliate. Calyx tube 1 (or less) 9
1.2–1.6 mm, puberulous outside; lobes ovate to oval,
1.5–2.8 9 1.2–1.4 mm, sparsely to densely pilose on both
sides, margins densely ciliate. Corolla tube infundibular,
1.5–3.5 9 2–2.5 mm on the top; lobes ovate to orbicular,
1–2 mm long and wide, glabrous or sparsely pubescent
outside, sparsely pilose on the base inside. Anthers
1–2 9 0.2–0.5 mm. Style 0.5–1 mm long; stigma elliptic,
1–2 9 0.3–0.5 mm, adaxially glabrous. Fruits globose,
10–12 9 10 mm, puberulous; stalk 3–5 mm long, pube-
rulous, bibracteate at the base. Seeds ovate to oblong,
2–3 9 2 mm.
Distribution and habitat: Luzon (Bataan, Batangas,
Bulacan, Pampanga, Pangasinan, Quezon, Tarlac, Zamb-
ales provinces and Manila); in low-altitude secondary
forest, on stream-sides, in shaded ravines, on clay or sandy
soils; 50–550 m asl (Fig. 5).
Local names: Bayabas-aso, Pagbut (Kpm.); Kasiway,
Sigay-sigay (Png.); Karagli, Kalagri, Malabayabas, Mal-
atadyang, Tulos-tulos (Tag.).
Phenology: Flowering January–November; fruiting
March–October.
Additional specimen examined: Luzon: Bataan Prov.,
Limay, Lamao, 14"30
0
N, 120"35
0
E, Nov. 1947, Edan
˜
o
4032 (L, NY); Lamao River, Mt. Mariveles, 14"26
0
N,
120"29
0
E, Feb. 1905, Meyer 2604 (US); Dinalupihan,
14"52
0
N, 120"27
0
E, Jan.–Feb. 1903, Merrill 1499 (US);
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 15
123
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without exact locality, Jun. 1917, de Guzman 26862 (US).
Batangas Prov., without exact locality, Aug. 1914, Ramos
1803 (L), Apr.–May 1915, Ramos & Deroy 22661 (K, US).
Bulacan Prov., Angat, 14"56
0
N, 121"02
0
E, Merrill 688 (L,
US), Merrill 223 (L, NY, US); Baliuag, 14"57
0
N,
120"53
0
E, Jan. 1910, Merrill 71 (US), Sep. 1913, Ramos
21724 (US), Ramos 21707 (L, US); Quingua, 14"53
0
N,
120"51
0
E, 12 May 1879, Vidal 1454(bis) (PNH). Manila,
Manila Bot. Garden, 14"34
0
N, 121"00
0
E, 1901, Ahern s.n.
(US \2 sheets[); Manila and vicinity, without exact
locality, Ramos 12182 (L). Pampanga Prov., Mt. Arayat,
15"09
0
N, 120 "45
0
E, Mar. 1903, Merrill 1379 (US), Mar.
1910, Curran 17738 (P, US); without exact locality,
Cuming 744 (L \2 sheets[). Pangasinan Prov., Alaminos,
16"09
0
N, 119"58
0
E, Dec. 1922, Mcgregor 41453 (K);
Bolinao, 16"19
0
N, 119"53
0
E, Jan. 2009, Adube & Alejandro
25076 (USTH), 25077 (PNH, USTH); Labrador, Mt. San
Isidro, 15"59
0
N, 120"06
0
E, Nov. 1917, Fenix 29978 (US);
Umingan, 15"54
0
N, 120"47
0
E, Apr.–Jun. 1912, Otanes
17748 (US); without exact locality, 1840, Callery s.n. 1
(P), Callery s.n. 2 (P). Tarlac Prov., Murcia, 15"24
0
N,
120"35
0
E, no collection date, Vidal 387 (A). Quezon Prov.,
Alabat Island, 14"60
0
N, 122"10
0
E, Sep.–Oct. 1926, Ramos
& Edan
˜
o 48182 (K, US). Zambales Prov., Subic, 14"53
0
N,
120"14
0
E, Apr. 1920, Edan
˜
o 38339 (US); without exact
locality, Jun. 9 1904, Maule 903 (US), Jan. 1 907, Curran
6066 (US), Nov.–Dec. 190 7, Ramos 4744 (US), Cuming
745 (L, NY).
Discussion: Villaria glomerata is distinguished by its
small flowers arranged in compact glomerules. As dis-
cussed above, the peduncles and pedicels of this species
continue to expand after anthesis. It is hermaphroditic as
are the other Villaria species. Repeated examination of the
flowers showed two stigmatic lobules, in contrast to the
earlier report of five lobules by Mulyaningsih and Ridsdale
(2004).
We regard the three sheets on which the type of Pla-
tymerium glomeratum, Haenke s.n., is actually mounted at
PR as one single collection. Therefore, we have not chosen
a single sheet to serve as a single lectotype.
For nearly all names authored by Blanco and published
in the ‘‘Flora de Filipina’’, no original type material ever
existed (cf. Merrill 1918, Nicol son and Arculus 2001).
Where neotypification of Blanco names becomes neces-
sary, Nicolson and Arculus (2001) recommended to rely on
the specimens cited as ‘‘illustrative specimens’’ by Merrill
(1918) in his ‘‘Species Blancoanae ’’. A so-called ‘‘first set’’
of ‘‘illustrative specimens’’ is housed at US, representing
the most complete and best documented set of all sets
distributed by Merrill. However, in the case of the four
synonymous names under V. glomerata that have been
identified already by Merrill, he only offers two ‘‘illustra-
tive specimens’’ for them in his Species Blancoanae,
namely nos. 223 and 688 (Merrill 1918: 364). We chose
them here as neotypes for Serissa pinnata, and Remijia
angatensis, respectively. We further selected one addi-
tional specimen housed at US, Merrill 71, to serve as
neotype for Serissa myrtifolia, and for the fourth name,
Remijia obscura, the specimen Vidal 387 (A) will serve as
another neotype.
4. Villaria leytensis Alejandro & Meve, sp. nov. TYPE:
Philippines, Visayas, Leyte, without exact locality, 30 Jun.
1915, Wenzel C.A. 1381 (holotype, A!; isotypes, G!, GH!,
US!).
Villaria leytensis folium forma atque magnitudine
folium Villaria acutifolia et morphologia floris Villaria
odorata simulans. Haec species ab omnis speciebus Vil-
laria lobis stigmaticis adaxialis papillatis et marginibus
foliorum sparsis ciliatis differt.
Shrubs or small trees; all vegetative parts glabrous
except for the sparsely cili ate leaf margins. Leaf blades
elliptic to ovate, 5–9.5 9
2.5–3.5 cm, coriaceous; apex and
base acute; secondary veins seven or eight pairs; petioles
3–5 mm long. Sti pules narrowly triangular, the keel
prominent from the tip along half of the length,
7–10 9 3–4 mm; colleters numerous, 0.5–0.75 mm long.
Inflorescences axillary, or supraaxillary 2 mm above the
node, three-flowered simple cymes, or uniflorous; pedun-
cles 2–2.5 cm long, glabrous. Flower pedicels 5–10 mm
long, glabrous; bracts ovate, apex rounded,
2–3 9 3–4 mm, glabrous outside, sparsely pubescent on
the base inside, margins sparsely ciliate. Calyx tube
4–5 9 3 mm, glabrous outside; lobes depressed ovate,
2 9 2.5–3 mm, glab rous outside, sparsely to densely pilose
at the base inside, margins sparsely ciliate. Cor olla tube
infundibular, 3–4 9 2.5–3 mm on the top; lobes oval to
orbicular, 4–4.5 9 4 mm, glabrous outside, densely pilose
on the base inside. Anthers 3 mm long, less than 1 mm
wide. Style 1.5–2 mm long; stigma narrowly ovate to
oblong, 3–4.5 mm long, 1–2 mm wide, adaxially papillate.
Fruits ellipsoid to globose, 9–11 9 8–9 mm, glabrous;
stalk 1–1.5 cm long, glabrous. Seeds ovate-oblong,
2.5–3 9 0.9–1.2 mm.
Distribution and habitat. Visayas (restr icted to Leyte).
Herbarium specimens lack information on the exact
locality and habitat of this species in Leyte. Probably also a
coastal species near the boarder of Leyte gulf (Fig. 5).
Local name: Unknown.
Phenology: Flowering March–June; fruiting June–
August.
Additional specimen examined: Visayas: Leyte, without
exact locality, 19 Jun. 1915, Wenzel C. A. 1363 (holotype,
US! \2 sheets[; isotypes: A! \2 sheets[, G! \2 sheets[,
NY!).
Discussion. Villaria leytensis resembles V. acutifolia in
the size and shape of leaves and V. odorata in the
16 G. J. D. Alejandro et al.
123
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morphology of flowers. This species can be distinguished
by having adaxially papillate stigmatic lobules and sparsely
ciliate leaf margins; neither feature was observed in any
other Villaria species. There is no habit information on this
plant in the herbarium collections, but considering other
Villaria species; we assum e that V. leytensis is a shrub to
small tree. This species is named after the Leyte province
of Visaya s, where the only known records (Wenzel 1363 &
Wenzel 1381) have been collected.
5. Villaria odorata (Blanco) Merr., Sp. Blancoan.:363.
1918; Enum. Philipp. Pl. 3:532. 1923. : Remijia odorata
Blanco, Fl. Filip. [F.M. Blanco] 2:115. 1845, 3:205. 1877.
TYPE: not designated. TYPE: Philippines, Luzon, Albay
Prov., Montufar [13"14
0
N 123"38
0
E], no collection date,
Vidal S. 3003 (neotype, designated here: K!; isoneotype,
A!). Fig. 3.
Gardenia elliptica Elmer, Leafl. Philipp. Bot. 1:6. 1906.
TYPE: Philippines, Luzon, Tayabas Prov., Atimonan,
August–September 1904, Whitford H. N. 705 (holotype,
PNH, destroyed; lectotype, designated here: NY!; isolec-
totypes, P!, US!).
Villaria littoralis S. Vidal, Phan. Cuming. Philipp.:180.
1885; Revis. Pl. Vasc. Filip.:154. 1886; Philipp. J. Sci.
3:437. 1908; Leafl. Philipp. Bot. 3:100 7. 1911. TYPE:
Philippines, Albay Prov., without exact locality or collec-
tion date, Cuming M. 874 (holotype, PNH, destroyed;
lectotype, designated here, P!).
Villaria rolfei S. Vidal, Phan. Cumi ng. Philipp.:180.
1885; Enum. Philipp. Pl. 3:532. 1923. : Randia rolfei (S.
Vidal) Hatus., Mem. Fac. Agric. Kagoshima Univ. 7:326.
1970. : Gardenia rolfei (S. Vidal) Hatus., Mem. Fac.
Agric. Kagoshima Univ. 5(3):50. 1966. TYPE: Philippines,
Albay Prov., without exact locality or date of collection,
Cuming M. 1271 (holotype, PNH, destroyed; lectotype,
designated here, L! \ 2 sheets [ ; isolectotypes, P!, G!).
Villaria philippinensis Rolfe, J. Linn. Soc., Bot. 21:311.
1884, Revis. Pl. Vasc. Filip.:154. 1886; Leafl. Philipp. Bot.
3:1007. 1911; Enum. Philipp. Pl. 3:532. 1923. TYPE:
Philippines, Luzon, Camarines Sur province, Minalabat?,
Jan. 1884, Vidal S. 836 (holotype, PNH, destroyed; lecto-
type, designated here, K!).
Shrubs or small trees, up to 6 m high; all vegetative
parts glabrous; lateral branches developing supraaxillary
in a distance of about 1 cm above the nodes. Leaf blades
vary from elliptic to ovate, oblong, or obovate, 8–13(–
20.5) 9 (3.5–)6–9 cm, coriaceous; apex varies from
broadly acute to acuminate, or obtuse; bases acute,
cuneate, or obtuse; secondary veins 7–12 pairs; petioles
5–10 mm long. Stipules triangular to ovate, keel entirely
prominent from the tip to the base, or only along
one-half to one-third of the length, 5–10 9 7–9 mm,
colleters numerous, 0.5–0.75 mm long, yellow to golden-
brown.
Inflorescences axillary, or supraaxillary 2–4 mm above
the nodes, usually three-flowered, simple or compound
dichasial cymes, rar ely uniflorous; peduncles 1–2(–6.5) cm
long, glabrous. Flower pedicels 5–7 mm long, glabrous;
bracts widely ovate, apex rounded, 3–4 9 4–5 mm, abax-
ially glabrous, adaxially sparsely pubescent at the base,
margins sparsely ciliate, som etimes with few scattered
colleters on the base inside. Calyx tube 3–4 mm long and
wide, glabrous outside; lobes depressed ovate,
2–2.5 9 3–4.5 mm, abaxially glabrous, adaxially sparsely
to densely pilose, o r only at the base, occasionally with few
colleters adaxially on the sides of the base, margins ciliate.
Corolla tube infundibular, 5–6 9 5 mm on the top; lobes
widely obovate to orbicular, 5–7 9 5–6 mm, abaxially
glabrous, adaxially sparsely pilose at the base. Anthers
5–5.5 9 1 mm. Style 2–3 mm long, stigma elliptic to
oblong, 5–6 9 1 mm, glabrous inside. Fruits ellipsoid to
globose, 14–16 9 10–14 mm, glabrous; stalk 0.5–2 cm
long, glabrous. Seeds ovate to oblong, 2–3 9 2 mm.
Distribution and habitat:. Villaria odorata is the most
widely distributed Villaria species occurring in Luzon
(Albay, Batanes, Bontoc, Bulacan, Cagayan, Isab ela,
Laguna, Mountain, Pampanga, Quezon, Rizal, and Sorso-
gon provinces), Visayas (Samar, Catarman), and Mindanao
(Siargao and Dinagat islands); in coastal forests, forest
margins, upper beaches, gully forest, or low- to mid-alti-
tude secondary forest; on clay or sandy soils; 0–1500 m asl
(Fig. 5).
Local names: Lanete, Lasgas (Bik.); Tango-tango
(BisC.); Botong (BisPn.); Tanhas (BisSL.); Otto (Ibg.);
Riki-riki (Ilk.); Butong-manok, Kumbatol (Tag.).
Phenology: Flowering February–August, and October–
December; fruiting January and March–November.
Additional specimens examined. Luzon: Aurora Prov.,
Baler, 15"46
0
N, 121"34
0
E, Aug.–Oct. 1903, Merrill 1027
(NY, US), Feb. 2010, Alejandro 25075 (A, K, L, NY, PNH,
US, USTH); Dibitugmin River, 15.42’N, 121"34
0
E, 06
May 1947, Quisumbing 2286 (A, PNH). Batanes Prov.,
Batan Island, Mahatao, 20"24
0
N, 121"57
0
E, May 1930,
Ramos 80402 (A); Jun.–Jul. 1930, Ramos 80601 (A); along
the road from Itbud to Mahatao (South to North), 20"22
0
N,
121"57
0
E, 26 Apr. 1996, Madulid & al. 23833 (K); Ivana,
20"21
0
N, 121 "55
0
E, May 1930, Ramos 79836 (A); without
exact locality, May–Jun. 1907, Fenix 3811 (NY, US). Bicol
Region, Camarines Norte, Paracale, 14"16
0
N, 122"46
0
E, 03
Mar 2007, de Leon 25056 (L, PNH, USTH), de Leon 25057
(A, NY, US, USTH), 29 Jul. 2007, Espino & al. 25051
(PNH, USTH), Espino & al. 25052 (PNH, USTH), Espino
& al. 25053 (L, USTH), Nov.–Dec. 1918, Ramos & Edan
˜
o
33655 (NY); Mt. Kadig, Minasag River, 14"12
0
N,
122"26
0
E, 21 Jun. 1959, Edan
˜
o 40436 (US); Mt. Bacacay,
14"13
0
N, 122"49
0
E, Nov.–Dec. 1918, Ramos & Edan
˜
o
33853 (US); Camarines Sur, Minalabac, 13"33
0
N,
Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) 17
123
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123"11
0
E, Sep.–Oct. 1958, Quisumbing 58330 (PNH),
Quisumbing 58331 (PNH), no collection date, Vidal 1454
(PNH \2 sheets[). Bontoc Prov., Mt. Masapilid, 17"05
0
N,
120"58
0
E, Mar. 1920, Ramos & Edan
˜
o 37887 (A, K, US).
Bulacan Prov., Quingua, 14"53
0
N, 120"51
0
E, Sep.–Oct.
1958, Quisumbing 58332 (PNH). Cagayan Prov., Pen
˜
a-
blanca, 17"37
0
N, 121"46
0
E, Apr. 1926, Ramos & Edan
˜
o
46576 (NY); Aparri, Bulala, vicinity of Pen
˜
ablanca,
18"22
0
N, 121"34
0
E, May–Jun. 1917, Add uru 7 (A, K, P,
US); Mt. Bawa, 18"18
0
N, 122 "05
0
E, Oct.–Nov. 1929, E-
dan
˜
o 78456 (A); Abulog, 18"26
0
N, 121 "26
0
E, 29 May
1917, Fenix 29235 (A, K, L, NY, P, US); Buguey, 18"16
0
N,
121"49
0
E, Apr. 1927, Clemens s.n. (NY); Malaueg (now
known as Rizal, Cagayan), 17"51
0
N, 121"21
0
E, Mar. 1886,
Vidal 3002 (K); Calayan, Brgy. Naguillan, So. Sisip,
18"55
0
N, 121"54
0
E, 12 Mar. 1996, Fuentes & Fernando
37135 (K); without exact localities, Feb. 1912, Ramos
13935 (P, US), Sep.–Oct. 1914, Velasco 23268 (US), May
1917, Pen
˜
as 26684 (US), Jul. 1923, Lizardo 29397 (A),
Mar. 1009, Darling 14742 (P). Calayan Island, Babuyanes,
19"19
0
N, 121"28
0
E, May 1917, Pen
˜
as 26714 (A, US).
Isabela Prov. Tumauini, 17"16
0
N, 121"52
0
E, no collection
date, Vidal 3004 (A, K); Palanan, So. Dimulid, 17"33
0
N,
122"25
0
E, 10 Jun. 2006, de Leon & Alejandro 25061 (K, A,
NY, PNH, USTH), de Leon & Alejandro 25062 (L, NY,
PNH, US, USTH), 11 Jun. 2006, de Leon & Alejandro
25063 (L, NY, USTH), de Leon & Alejandro 25064 (K, L,
A, US, USTH); Divilacan municipality, Sobok Point,
17"14
0
N, 122"25
0
E, 22 May 1991, Co 3532 (L, NY), Co
3537 (A, CAHUP, US); Dicotkotan, Brgy. San Isidro,
17"08
0
N, 122"30
0
E, 18 May 1992, Co 61636 (CAHUP), 05
Apr. 1991, Gaerlan & al. 2923 (K); Palanan Bay, 16"58
0
N,
122"26
0
E, Jun. 1913, Escritor 21152 (US), Escritor 21163
(P, US); without exact locality, May 1909, Ramos 8080
(US). Laguna Prov., Los Ban
˜
os, 14"09
0
N, 121"13
0
E, Apr.
1910, Tamesis 11933 (NY, P, US); Mt. Makiling, 14"07
0
N,
121"12
0
E, 12 Aug. 1946, Lorenzo 9754 (PNH); UPLB,
Forestry arboret um, 14"08
0
N, 121"11
0
E, 27 Mar. 1954,
Lagrimas 2303 (CAHUP \2 sheets[), 18 Aug. 1945,
Lorenzo 174 (CAHUP\2 sheets[), 28 Feb. 1953, Dequilia
1709 (CAHUP \2 sheets[). Mindoro Island, Mt. Halcon,
Rambiao River, 13"15
0
N, 120"59
0
E, Jan.–Feb. 1948, Edan
˜
o
3511 (A, PNH). Mount ain Prov., Tanit, 17"05
0
N, 121"10
0
E,
Aug. 1923, Lizardo 29381 (A). Pampanga Prov., Sicorsico,
Baso, Arayat, Mt. Arayat, 15"12
0
N, 120"43
0
E, 09 Feb.
1992, Gaerlan & al. 10266 (K), 09 May 1992, Gaerlan &
al. 10318 (K). Quezon Prov., General Nakar, 14"45
0
N,
121"37
0
E, 03 Mar. 2006, de Leon & Alejandro 25058 (K,
L, NY, USTH), 20 May 2006, de Leon & Alejandro 25055
(A, K, USTH), 27 Apr. 2007, Espino & al. 25054 (NY, US,
USTH); Mauban, 14"10
0
N, 121"43
0
E, Mar. 1908, Curran
10183 (US), 17 Jan. 1913, Ramos 19488 (K), Jun.–Jul.
1916, Cailipan 26013 (A, P), Oct. 1918, Quieb 27396 (A,
K, P, US), Dec. 2005, de Leon & Alejandro 25059 (A, L,
US, NY, USTH, PNH), de Leon & Alejandro 25060 (K, L,
PNH, USTH), de Leon & Alejandro 25065 (L, NY, PNH,
USTH), de Leon & Alejandro 25066 (A, US, K), de Leon &
Alejandro 25067(L, NY, US, USTH), de Leon & Alejandro
25068 (A, K, PNH, USTH), 11 Mar. 2007, de Leon &
Alejandro 25069 (L, NY, PNH, USTH), de Leon & Alej-
andro 25070 (A, G, K, US, USTH), de Leon & Alejandro
25071 (L, NY, PNH, USTH), de Leon & Alejandro 25072
(A, G, K, US, USTH), de Leon & Alejandro 25073 (A, K,
L, NY, US, PNH, USTH), de Leon & Alejandro 25074
(A,G, K, L, US, NY, PNH, USTH); Polillo Island, 14"50
0
N,
121"57
0
E, May–Jun. 1924, Salvoza 29637 (NY); Tignoan,
Infanta, 14.44
0
N, 121"39
0
E, Sep. 1904, Whitford 844 (P,
US), Whitford 752 (P), 08 Feb. 1956, Lagrimas 37269 (L,
NY, PNH \2 sheets[); Tayabas, Laguimanoc (now known
as Padre Burgos), 13.56’N, 121" 48
0
E, Sep.–Oct. 1958,
Quisumbing 58341 (PNH), no collection date, Vidal
1467(d) (A \2 sheets[, K, PNH), Guinayangan, 13"54
0
N,
122"27
0
E, Mar.–Apr. 1913, Escritor 20772 (P, US), Escr-
itor 20797 (L, NY, P, US); Real, Tignoan, 14"39
0
N,
121"36
0
E, 08 Mar. 1956, Lagrimas s.n. (PNH); Alabat
Island, 14"06
0
N, 122"01
0
E, Sep.–Oct. 1926, Ramos &
Edan
˜
o 48185 (A, NY), Brgy. Bacung, Mt. Camagon,
14"09
0
N, 122"02
0
E, 03 Jan. 1996, Gaerlan & Romero
23296 (K). Rizal Prov., Feb. 1913, Loher 14207 (A).
Sorsogon Prov., Irosin, Mt. Bulusan, 12"46
0
N, 124"03
0
E,
Jul. 1916, Elmer 16770 (A \2 sheets[, G, K, NY, P, US
\2 sheets[); Bulusan, Cabugawan River, 12"44
0
N,
124"08
0
E, 21 Jun. 1958, Sinclair &. Edan
˜
o 9687 (K).
Visayas: Samar, Catarman, 12"29
0
N, 124"38
0
E, Apr. 1909,
Rosenbluth 12641 (P, US), Capul, 12"24
0
N, 124"10
0
E, 27
Mar. 1957, Kondo & Edan
˜
o 36798 (K, PNH \2 sheets[).
Mindanao: Surigao del Norte, Siargao Island, without
exact locality, Jun. 1919, Ramos & Pascasio 34950 (NY),
Ramos & Pascasio 34978 (L, NY); Dinagat Island,
09"40
0
N, 125"38
0
E, 1902, Ahern 457 (US \2 sheets[);
Catel, 05 Oct. 1906, Merrill 5441 (NY, US). No locality,
Cuming s.n. (P).
Discussion: Previously, the characters used to recognize
the four Villaria species (V. littoralis, V. odorata, V. phi-
lippinensis and V. rolfei) were mainly based on slight dif-
ferences in the shape and size of leaves, stipules, and
inflorescences. However, close simi larities between these
species were already noted by Rolfe (1884), Vidal (1885),
and Merrill (1910, 1918, 1923). Based on further compara-
tive morphological study, we conclude that the distinctive
features of these species are rather weak. The vegetative and
reproductive morphology of these taxa are more or less
identical. They only vary in the overlapping shape of leaves
and length of stipules and inflorescences. Therefore, these
four Villaria species are considered here as conspecific.
Following the ICBN, V. odorata has priority.
18 G. J. D. Alejandro et al.
123
Author's personal copy

Some taxonomic conflict was raised by Fernandez-Vil-
lar (1880) who synonymized the basionym of V. odorata,
Remijia odorata Blanco, under Randia densiflora (Wall.)
Benth., a vegetatively similar Asian species now mostly
treated as Aidia densiflora (Wall.) Masam. However, the
latter species is a larger tree of montaneous forests bearing
small flowers with much exserted stigmas. Although
Merrill (1918) already realized that F.-Villar was mistaken,
the wrong synonymy has been repeated in the Rubiaceae
checklist of Kew (Govaerts et al. 2006).
The type material of Blanco could not be traced, and it is
not reported whether the specimen that was used for the
production of the full colour plate (chromolithography) in
the Atlas I of the third edition of Flora Filipina (Naves
1882: t. 56) belonged to Blanco’s original material, or to
additional material collected by A. Naves or his collabo-
rator C. Fernandez-Villar after Blanco’s death. Plate 56
could represent a lecto(icono)type, however, and because
of the doubtful origin of the specimen depicted we selected
one of S. Vidal’s collection from Luzon, Albay province
(Vidal 3003), as neotype. The neotype specimen selected
from K possesses a flowering and a fruiting branch.
Acknowledgments We thank the following herbaria and their staff
for providing loans and/or access to collections: A, CAHUP, G, GH, K,
L, NY, P, PNH, PR, US and USTH. We are also grateful to three
anonymous reviewers for helpful comments and suggestions. The first
author thanks Angelika Ta
¨
uber for guidance during the molecular work,
Irma Mika for Latin translations, Nemecio Diego for some parts of the
illustrations, the University of Santo Tomas, Manila, Philippines, for
some financial support during the field collection; and the Alexander
von Humboldt Foundation for a Postdoctoral Fellowship grant.
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