Plant Syst Evol (2009) 278:101–123
DOI 10.1007/s00606-008-0138-4
ORIGINAL ARTICLE
Deep divergences in the coffee family and the systematic position
of Acranthera
Catarina Rydin Æ Kent Kainulainen Æ
Sylvain G Razafimandimbison Æ Jenny E E Smedmark Æ
Birgitta Bremer
Received: 31 March 2008 / Accepted: 28 November 2008 / Published online: 12 February 2009
Ó Springer-Verlag 2009
Abstract Despite extensive efforts, there are unresolved
questions on evolutionary relationships in the angiosperm
family Rubiaceae. Here, information from six loci and 149
Rubiaceae taxa provide new insights. Acranthera and
Coptosapelta are strongly supported as sisters. Pollen grains
of Acranthera possess several features common in Rubiaceae, but amongst potential similarities with the unusual
grains of Coptosapelta are the nature of the apertures and
the structure of the sexine. Luculia, Acranthera and
Coptosapelta are excluded from the three subfamilies
Ixoroideae, Cinchonoideae and Rubioideae. Sipaneeae and
Condamineeae form a clade, sister to remaining Ixoroideae.
Rondeletieae and Guettardeae are sisters to remaining
Cinchonoideae. Colletoecema is sister to remaining
Rubioideae, followed by the Urophylleae–Ophiorrhizeae
clade. Nuclear ITS provided structured information at all
phylogenetic levels, but the main gain from adding nrITS
was the increased resolution. Average support values also
increased but were generally high also without nrITS and
the increase was not statistically significant.
Keywords Anther-stigma complex Cinchonoideae
Coptosapelta Ixoroideae Luculia Rubioideae
Introduction
Rubiaceae is one of the largest families of flowering plants,
comprising more than 13,000 species (Govaerts et al.
2006). Distribution is worldwide, with a particularly high
diversity in the tropics and subtropics. The family is a welldefined monophyletic group that can be easily recognised
by (generally) opposite branching and phyllotaxis, interpetiolar stipules and sympetalous and epigynous flowers
(Schumann 1891; Hutchinson 1973).
Phylogenetic studies generally recognise three major
lineages within Rubiaceae (Bremer et al. 1995; Bremer
1996b, 1999; Rova et al. 2002; Robbrecht and Manen 2006),
often referred to as subfamilies Rubioideae, Ixoroideae and
Cinchonoideae sensu Bremer et al. (1999). Subsequent
studies have further investigated relationships within these
subfamilies, for example Bremer and Manen (2000,
Rubioideae), Andreasen and Bremer (2000, Ixoroideae) and
Andersson and Antonelli (2005, Cinchonoideae).
However, despite these extensive efforts, several questions on evolutionary relationships within Rubiaceae,
including deep divergences in the family, have remained
unanswered. We introduce some of the unresolved questions here.
Acranthera
C. Rydin
Institute of Systematic Botany, University of Zürich,
Zollikerstrasse 107, 8008 Zürich, Switzerland
C. Rydin (&) K. Kainulainen S. G. Razafimandimbison
J. E. E. Smedmark B. Bremer
Department of Botany, Bergius Foundation, Royal Swedish
Academy of Sciences, Stockholm University,
106 91 Stockholm, Sweden
e-mail: Catarina.Rydin@botan.su.se
Acranthera (Arnott 1838) is distributed in India, South to
Central China and Central Malesia and consists of about 40
species of sparsely branched subshrubs (Bremekamp 1947;
Govaerts et al. 2006). The flowers of Acranthera are
unique within Rubiaceae and are characterised by the
presence of united connective appendages, which in turn
are united with the stigma by means of a columnar tissue
(Puff et al. 1995). In the original description, Arnott (1838)
123
102
made a remark on a possible affinity to Mussaenda [now
placed in the tribe Mussaendeae sensu Bremer and Thulin
(1998) of Ixoroideae]. Bremekamp (1947) questioned this
affinity in his monograph of the genus and considered the
position of Acranthera unknown. He later classified
Acranthera as a monogeneric tribe within Ixoroideae
(Bremekamp 1966).
Since then, only a few studies have investigated this
genus. Puff et al. (1995) described the pollination ecology,
morphology and anatomy of the stamens in selected
Acranthera species and Kiehn (1995) included Acranthera
in a survey of chromosome data. Furthermore, Acranthera
was assigned to the tribe Sabiceeae (Cinchonoideae sensu
Bremekamp 1966) based on the results of a morphologicalbased phylogenetic study by Andersson (1996). Bremer
and Thulin (1998) did not include Acranthera in their
molecular study but argued that its testa structure is different from that of Sabicea, being instead similar to that of
Amphidasya. They postulated on a possible placement of
Acranthera in Rubioideae.
One paper has addressed the phylogenetic position of
Acranthera based on molecular data; Alejandro et al.
(2005) analysed trnT–L–F chloroplast data and the genus
was resolved as sister to the rest of subfamily Rubioideae,
with a relatively high statistical support. This study
focused, however, on Mussaenda and allied genera.
Luculia (Rubiaceae) was used as outgroup, and the sampling within Rubioideae and Cinchonoideae was limited.
Coptosapelta and Luculia
Coptosapelta consists of 16 species from South East Asia
(Valeton 1923; Govaerts et al. 2006). They are woody vines
with axillary, pentamerous flowers (Chao 1978). The genus
was originally described by Korthals (1851) and placed in
the tribe Cinchoneae (subfamily Cinchonoideae), but the
morphology and phylogeny of the genus were later reinvestigated and debated by many authors (e.g. Verdcourt
1958; Bremekamp 1966; Robbrecht 1988; Andersson and
Persson 1991). Bremekamp (1952, 1966) recognised the
tribe Coptosapelteae in subfamily Ixoroideae.
Luculia comprises four species of trees or shrubs with
showy flowers, distributed in Himalaya, northern Thailand
and southern China (Polunin and Stainton 1984; Govaerts
et al. 2006). Luculia was also placed in Cinchoneae by
Schumann (1891), and subsequent authors (e.g. Verdcourt
1958; Bremekamp 1966; Robbrecht 1988) did not
disagree. Based on the phylogenetic analysis of morphological data, Andersson and Persson (1991) included
Luculia and several other genera in a much wider circumscription of Coptosapelteae, which was later shown to
be highly polyphyletic (Razafimandimbison and Bremer
2001).
123
C. Rydin et al.
In phylogenetic studies based on molecular data, Luculia
and Coptosapelta have typically had isolated, unresolved or
poorly supported positions, often amongst the basal nodes
within the family (Bremer and Jansen 1991; Bremer et al.
1995; Bremer 1996b; Bremer et al. 1999; Bremer and
Manen 2000; Rova et al. 2002; Robbrecht and Manen 2006).
The diversity of results that has been presented indicates that
the position(s) of Luculia and Coptosapelta is not confidently resolved. The two genera form a clade in some
studies (e.g. Andersson and Persson 1991; Robbrecht and
Manen 2006), in others they appear more distantly related to
each other (Bremer et al. 1999). In some studies, Luculia is
sister to remaining Rubiaceae (Bremer and Jansen 1991,
Coptosapelta not included), in others they are sister to
Cinchonoideae–Ixoroideae (Robbrecht and Manen 2006).
Urophylleae and Ophiorrhizeae
The subfamily Rubioideae was proposed by Bremekamp
(1952), based e.g. on the presence of raphide idioblasts, and
was formally described by Verdcourt (1958). Andersson
and Rova (1999) and Bremer and Manen (2000) addressed
the phylogeny of the subfamily but some results were
poorly supported and/or differed between the studies. For
example, Andersson and Rova (1999) found a sister relationship between Urophylleae and Ophiorrhiza, this clade
being the sister of remaining Rubioideae. Bremer and
Manen (2000), who used a larger sample of species and
more characters, found a basal grade within Rubioideae,
with Ophiorrhizeae as the earliest diverging clade, followed
by Urophylleae and Lasiantheae.
Aims of this study
After more than 60 phylogenetic studies during the last
18 years (adjusted from Bremer in press) many aspects of
Rubiaceae evolution are now relatively well understood.
There are, however, phylogenetic questions that remain
unanswered, which hampers further studies addressing for
example biogeography and geographical origin, molecular
dating of divergences, ancestral state reconstruction and
character evolution within the family. We address deep
divergences in Rubiaceae with special emphasis on
Acranthera, and we investigate the usefulness of nrITS for
analysing deep divergences in Rubiaceae.
Materials and methods
Selection of species and laboratory procedures
We selected 149 taxa for the present study (Table 1), representing the major clades within Rubiaceae. We included
Deep divergences in the coffee family and the systematic position of Acranthera
85 terminals (representing 16 tribes) from Rubioideae,
26 terminals (representing 13 tribes) of Ixoroideae, 11 terminals (representing 7 tribes) of Cinchonoideae, and in
addition seven terminals of Acranthera, eight of Coptosapelta and four of Luculia. Eight outgroup taxa from the
sister group of Rubiaceae (the other families within Gentianales, Backlund et al. 2000) were selected and sampled at
the generic level. Ingroup sequences were sampled at the
species level.
We utilised information from six loci: five chloroplast
regions (rbcL, rps16 intron, ndhF, atpB–rbcL spacer,
trnT–L–F region) and the internal transcribed spacer of
the nuclear ribosomal DNA (nrITS1, 5.8S, nrITS2). We
used sequences from GenBank whenever available and
we also produced 249 new sequences for this study.
GenBank accession numbers are given in Table 1. DNA
was extracted, amplified and sequenced using standard
procedures outlined in Kårehed and Bremer (2007).
References to primers are given in Table 2. Sequence
fragments were assembled using the Staden package
(Staden 1996).
Alignment
Alignments of rbcL, rps16, ndhF, atpB–rbcL spacer and
trnT–L–F could easily be performed by eye using the
software Se–Al v.2.0 (Rambaut 1996). Insertion/deletion
events were visually inferred, following the alignment
criteria outlined in Oxelman et al. (1997). Gaps were
treated as missing data in the alignment and added as
binominal characters (absent or present) at the end of the
matrix.
In order to investigate if nrITS could be utilised for
investigating deep divergences in Rubiaceae, we performed
an initial alignment using Clustalx/Clustalw (Chenna et al.
2003). From the resulting alignment, it was obvious that
most of the region could very easily be aligned over the
entire family. Two short regions, one located in nrITS1, the
other in nrITS2, were not properly aligned in Clustal and
we edited the output from Clustal by eye. We made a
simple parsimony analysis to evaluate the amount of
information in nrITS. The resulting tree was partly collapsed in basal parts, but added valuable information on
higher-level relationships. We continued by adding nrITS
to the combined data set and compared results from bootstrap analyses including and excluding nrITS. We further
conducted a bootstrap analysis on the combined six-gene
data set where we removed the two regions (mentioned
above), which were more difficult to align. Parts removed
correspond to positions 173-236 and 537-541 in the nrITS
sequence of Luculia gratissima (GenBank accession:
EU145344).
103
Phylogenetic reconstruction
We analysed each gene separately, including and excluding
information from indels. In order to evaluate the usefulness
of nrITS, we performed combined analyses including and
excluding nrITS (5-cp data set; six-gene data set). We
further analysed the combined six-gene data set, including
and excluding information from indels. All matrices were
analysed with two approaches: Bayesian inference and
parsimony.
Bayesian analyses were performed in MrBayes
3.1 (Huelsenbeck and Ronquist 2001; Ronquist and
Huelsenbeck 2003). For each single gene data set, the best
performing evolutionary model was identified under three
different model selection criteria: Akaike information
criterion (AIC) (Akaike 1973), AICc (a second order AIC,
necessary for small samples) and the Bayesian information
criterion (BIC) (Schwartz 1978). We performed these
calculations in software MrAIC ver. 1.4.3 (Nylander 2004).
Indels were treated as a morphological partition.
For single gene analyses, one million generations were
run, with a sample frequency of 1,000 and four parallel
chains. Prior probabilities were specified as follows
(according to output from MrAIC): a flat Dirichlet prior
probability (all values set to 1.0) was selected for the
substitution rates (revmatpr) and the nucleotide frequencies
(statefreqpr). The prior probability for the shape parameter
of the gamma distribution of rate variation (shapepr) was
uniformly distributed in the interval (0.1, 50.0). For analyses using a gamma distribution with a proportion of
invariable sites, we specified a prior probability for this
proportion (pinvarpr), uniformly distributed on the interval
(0.0, 1.0).
For combined analyses, five million generations were
run. We partitioned the combined data set in several ways.
First, we included all sequence data into a single partition
and analysed it together with the morphological partition.
Second, we included all chloroplast regions in one partition, and specified a separate partition for the nuclear
ribosomal ITS. Indels constituted a separate morphological
partition as before. We further excluded gap coding
information and partitioned the molecular data into two
partitions: chloroplast data and nrITS data. Finally, we
specified seven partitions, one for each gene region, and
one for indels. In all analyses, partitions were unlinked so
that each partition was allowed to have its own set of
parameters. Convergence of runs was assessed from the
average standard deviation of split frequencies, chain swap
information and potential scale reduction factors.
To investigate the usefulness of nrITS in the present
study, we performed further analyses on the combined data
set, (1) excluding nrITS, and (2) excluding potentially
123
104
123
Table 1 The data matrix
Taxon
Voucher (of previously
unpublished sequences)
Acranthera Arn. ex Meisn. (sp. 1) Ridsdale 2470 (L)
Classification
rbcL
–Coptosapelteae
AM11719835 EU145477*
rps16
35
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
EU145400*
–
AJ84740842
–
Acranthera Arn. ex Meisn. (sp. 2) Bremer 1731 (UPS)
–Coptosapelteae
AM117199
EU145478*
–
EU145312*
EU145524*
–
Acranthera atropella Stapf
KH Kjeldsen 54 (AAU)
–Coptosapelteae
–
EU145480*
–
–
EU145525*
–
Acranthera frutescens Valeton
AD Poulsen 52 (AAU)
–Coptosapelteae
EU145449*
EU145475*
EU145398*
EU145310*
EU145522*
EU145345*
Acranthera grandiflora Bedd.
Klackenberg & Lundin 541 (S) –Coptosapelteae
EU145448*
EU145474*
EU145397*
EU145309*
EU145521*
–
Acranthera siamensis (Kerr)
Bremek.
Larsen 45665 (AAU)
–Coptosapelteae
EU145450*
EU145476*
EU145399*
EU145311*
EU145523*
EU145346*
Acranthera siamensis(?) (Kerr)
Bremek.
Puff 990826-1/1 (WU)
–Coptosapelteae
AM11720035 EU145479*
EU145401*
EU145313*
–
–
IXO-Gardenieae
Z6884419
AF20097429
–
–
AF20102829
AJ22483517
Tonkin 200 (UPS)36
IXO-Alberteae
Y1870817
EU145491*
AJ23628216
–
AJ62011847
AJ22484217
Clark & Watt 736 (UPS)
APOCYNACEAE
RUB-Urophylleae
X917607
Y1184414
AJ4310324
AF12927124
AJ0119825
–
DQ3591616
EU145337*
AJ4309074
EU145576*
DQ3588806
EU145383*
GENTIANACEAE
L143898
–
AJ2358299
DQ1316956
AJ49019044
AJ48986444
Aidia micrantha (K.Schum.)
Bullock ex F. White
Alberta magna E. Mey.
Alstonia scholaris (L.) R.Br.
Amphidasya ambigua (Standl.)
Standl.
Anthocleista Afzel. ex R.Br.
Anthospermum herbaceum L.f.
Bremer, 3093 (UPS)
Arcytophyllum aristatum Standl.
Argostemma hookeri King
Malaysia, Wanntorp s.n. (S)
1
16
RUB-Anthospermeae
X83623
EU145496*
AJ236284
RUB-Spermacoceae
AJ2885952
AF33334820
–
–
RUB-Argostemmateae
Z6878821
EU145497*
EU145419*
AJ2340322
EU145545*
EU145356*
–
–
AM26690237
AM26698937
2
AM266813
37
AJ234028
2
EU145544*
EU145355*
AF33334920
AM18206157
Batopedina pulvinellata Robbr.
RUB-Knoxieae
AJ288596
Bertiera guianensis Aubl.
IXO-Bertiereae
AJ22484517
AF20098329
–
–
AF15267012
AJ22484117
Bouvardia ternifolia (Cav.)
Schltdl.) (syn. Bouvardia
glaberrima)
RUB-Spermacoceae
X836261
AF00275811
–
X7647841
DQ3591656
DQ3588846
Calycophyllum candidissimum
(Vahl) DC.
IXO-Condamineeae
X836271
AF00403011
AJ23628516
DQ1317086
AF15264612
DQ3588866
Dirichletia glaucescens Hiern
(syn. Carphalea glaucescens)
Catesbaea spinosa L.
RUB-Knoxieae
Z6878921
AM26681737 AJ23628716
–
AM26690637
AM26699337
CINCH-Chiococceae
X836281
AF00403211
AF15270612
AY76388013
1
11
Cephalanthus occidentalis L.
CINCH-Naucleeae
X83629
Chiococca alba (L.) Hitchc.
CINCH-Chiococceae
L143948
AF00403411
CINCH-Chiococceae
1
11
Cinchona pubescens Vahl
Coccocypselum condalia Pers.
Pirani & Bremer 4891 (SPF)
X83630
AF004033
AF004035
35
AM11734335 –
AJ236288
16
AJ13083516
AJ235843
9
DQ131710
6
DQ1317116
AJ233990
2
AF152692
12
AY76381313
AJ346963
3
AJ3468833
DQ3588876
AY53835615
AM117217
EU145499*
EU145420*
EU145324*
EU145547*
EU145358*
RUB-Coussareeae
X8714522
EU145500*
EU145421*
EU145325*
EU145548*
EU145359*
Coffea arabica L.
IXO-Coffeeae
X836311
AF00403811
AJ23629016
X7036440
DQ15384546
DQ15360946
C. Rydin et al.
RUB-Coussareeae
Coccocypselum hirsutum Bartl. ex CT 908, Bremer 2700 (S)
DC.
Taxon
Voucher (of previously
unpublished sequences)
Classification
rbcL
trnT/F
nrITS
Colletoecema dewevrei (De Wild.) S Lisowski 47195 (K)
E.M.A. Petit
RUB-Colletoecemateae
EU14545761 AF12927224
EU14540961 DQ1317136
EU14553261
EU145353*
Condaminea corymbosa (Ruiz &
Pav.) DC.
IXO-Condamineeae
Y1871316
AF00403911
AJ23629116
–
AF10240643
–
rps16
ndhF
atpB-rbcL
spacer
Coptosapelta diffusa (Champ.)
Steenis (specimen 1)
Bartholomew et al. 847 (AAU) –Coptosapelteae
EU145452*
EU145482*
EU145403*
EU145315*
EU145527*
EU145347*
Coptosapelta diffusa (Champ.)
Steenis (specimen 2)
Steward et al. 594 (S)
–Coptosapelteae
EU145453*
EU145483*
EU145404*
AJ2339872
DQ3591666
DQ3588826
Coptosapelta flavescens Korth.
(specimen 1)
Puff 950720-1/2 (WU)
–Coptosapelteae
Y1871416
EU145484*
AJ23629216
EU145316*
AM11735435
EU145348*
Coptosapelta flavescens Korth.
(specimen 2)
Gardette et al. EG1716 (K)
–Coptosapelteae
EU145454*
EU145485*
EU145405*
EU145317*
EU145528*
EU145349*
Coptosapelta flavescens Korth.
(specimen 3)
Larsen et al. 31147 (AAU)
–Coptosapelteae
–
EU145488*
EU145408*
–
EU145531*
EU145352*
Coptosapelta montana Korth. ex
Valeton
Clemens & Clemens 40864 (K) –Coptosapelteae
EU145451*
EU145481*
EU145402*
EU145314*
EU145526*
–
Coptosapelta tomentosa Valeton
ex K.Heyne (specimen 1)
Beusekom & Charoenpol 1741 –Coptosapelteae
(AAU)
EU145455*
EU145486*
EU145406*
EU145318*
EU145529*
EU145350*
Coptosapelta tomentosa Valeton
ex K.Heyne (specimen 2)
Beusekom & Charoenpol 1933 –Coptosapelteae
(AAU)
EU145456*
EU145487*
EU145407*
EU145319*
EU145530*
EU145351*
Coussarea hydrangeifolia (Benth.) Fuentes 5504 (GB)
Benth. & Hook.f. ex Müll. Arg.
RUB-Coussareeae
EU145460*
EU145501*
EU145422*
EU145326*
EU145549*
EU145360*
Coussarea macrophylla (Mart.)
Müll. Arg.
RUB-Coussareeae
Y1184714
AF00404011
–
–
AF15261212
(C. sp)
–
Cremaspora triflora (Thonn.)
K.Schum.
IXO-Cremasporeae
Z6885619
AF20099029
–
DQ1317186
AF20104029
AJ22482417
–
AM11735635
–
EU145550*
–
AF15270112
AY76389113
AJ2340152
–
AY51406153
AJ2340192
DQ66213832
EU145364*
Cremocarpon lantzii Bremek.
Razafimandimbison 517 (UPS) RUB-Psychotrieae
AM11722235 AM11729635 –
2
–
X836321
AF00404411
AM11734535 DQ1317206
RUB-Morindeae
Z6879321
AF33164720
–
Danais xanthorrhoea (K. Schum.) Bremer 3079 (UPS)
Bremek.
RUB-Danaideae
Z6879421
AM11729735 AJ23629316
Declieuxia cordigera Mart. &
Zucc. ex Schult. & Schult.f.
Pirani & Bremer 4893 (SPF)
RUB-Coussareeae
AM11722435 AM11729835 EU145423*
EU145327*
EU145551*
EU145361*
Declieuxia fruticosa (Willd. ex
Roem. & Schult.) Kuntze
B. Hammel 20875 (MO, CR)
RUB-Coussareeae
AJ00217723
DQ1317216
EU145552*
EU145362*
Rodriguez 10 (K)
RUB-Coussareeae
AJ288599
Cubanola domingensis (Britton)
Aiello
CINCH-Chiococceae
Damnacanthus indicus C.F.
Gaertn.
123
EU145503*
–
105
EU145502*
Cruckshanksia hymenodon Hook.
& Arn.
AJ234004
2
Deep divergences in the coffee family and the systematic position of Acranthera
Table 1 continued
106
123
Table 1 continued
Taxon
Voucher (of previously
unpublished sequences)
Dentella repens (L.) J.R.Forst. &
G.Forst.
Classification
rbcL
rps16
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
RUB-Spermacoceae
–
AF33337020
–
–
AF38154049
–
Dibrachionostylus kaessneri
(S.Moore) Bremek.
Strid 2564 (UPS)
RUB-Spermacoceae
AJ61621128
AF00276111
–
–
EU145574*
–
Didymaea alsinoides (Cham. &
Schltdl.) Standl.
Keller 1901 (CAS)
RUB-Rubieae
Z6879521
–
–
AJ2340362
EU145570*
–
Diplospora polysperma Valeton
Ridsdale IV.E.130 (L)
IXO-Coffeeae
AJ28670318
–
AM11730135 –
EU145538*
–
Dunnia sinensis Tutcher
(Specimen 1)
Yangchun 10, Ge et al. 2002
RUB-Dunnieae
EU145467
EU14551561 EU14544261 EU14533961
EU14558361
EU145390*
Dunnia sinensis Tutcher
(Specimen 2)
Taishan 10, Ge et al. 2002
RUB-Dunnieae
EU14546861 EU14551661 EU14544361 EU14534061
EU14558461
EU145391*
Dunnia sinensis Tutcher
(Specimen 3)
Zhuhai 12, Ge et al. 2002
RUB-Dunnieae
EU14546961 EU14551761 EU14544461 EU14534161
EU14558561
EU145392*
Dunnia sinensis Tutcher
(Specimen 4)
Longmen 12, Ge et al. 2002
RUB-Dunnieae
EU14547061 EU14551861 EU14544561 EU14534261
EU14558661
EU145393*
Dunnia sinensis Tutcher
(Specimen 5)
Xinhui 16, Ge et al. 2002
RUB-Dunnieae
EU14547161 EU14551961 EU14544661 EU14534361
EU14558761
EU145394*
IXO-Condamineeae
Y1871516
AF15263712
–
Emmenopterys henryi Oliv.
Unknown Rubiaceae (GenBank
name: Ernodea littoralis Sw.)
Faramea multiflora A Rich.
Bremer et al. 3331 (UPS)
61
RUB-Spermacoceae
AJ288601
RUB-Coussareeae
Z6879621
AM11730235 AJ23629416
2
AF002763
11
AF00404811
35
35
DQ1317286
2
–
AJ234025
–
EU145424*
EU145328*
AF10242243
EU145412*
6
EU145534*
–
–
EU145363*
Ferdinandusa speciosa Pohl
Malme 2442 (UPS)
IXO-Condamineeae
AM117226
Feretia aeruginescens Stapf
Bremer 3137 (UPS)
IXO-Octotropideae
Z6885719
AM11730535 –
–
EU145539*
Fernelia buxifolia Lam.
de Block s.n. (BR)
IXO-Octotropideae
AJ28670418
AM11730635 –
DQ1317366
EU145540*
–
–
X7645941
–
–
AJ0119845
L3640038
AJ2339852
AF10242843
39
DQ398604
X7789345
DQ3588816
DQ39863939
–
–
EU145569*
–
EU145533*
AY73029430
AJ84740742
–
AF38153949
–
27
AM117304
11
Galium album Mill.
RUB-Rubieae
X81090
Gelsemium Juss.
Gentiana L.
GELSEMIACEAE
GENTIANACEAE
L143978
L143988
RUB-Psychotrieae
AM11722835 AF36984526
Geophila obvallata Didr.
Guettarda uruguensis Cham. &
Schltdl.
Gynochthodes coriacea Blume
Hedyotis fruticosa L.
Q Luke 9037 (FR)
X5-127, Gillis 9575 (FTG)
1
CINCH-Guettardeae
X83638
RUB-Morindeae?
AJ2886032
RUB-Spermacoceae
21
Z68799
1
CINCH-Hillieae
X83642
Houstonia caerulea L.
RUB-Spermacoceae
AJ2886042
AJ4310334
AJ4310344
EU145489*
AJ236297
16
AM11731135 –
–
–
–
AM117315
35
AF33337920
AJ236298
–
DQ131739
6
16
AJ234026
2
AJ233993
2
AM117362
35
AF38152449
–
–
DQ01270658
DQ01277458
Hydnophytum formicarum Jack
RUB-Psychotrieae
X83645
1
AF001339
11
–
X76480
41
–
AF03491223
C. Rydin et al.
Hillia triflora (Oerst.) C.M. Taylor
AF004050
DQ131735
Taxon
Voucher (of previously
unpublished sequences)
Classification
rbcL
rps16
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
Hymenodictyon floribundum
(Hochst. & Steud.) Rob.
Puff 861109-3/1 (WU)
CINCHHymenodictyoneae
AJ3470153
AF00405811
EU145411*
DQ1317426
AY53845415
AJ3469053
IXO-Ixoreae
X836461
AM11732135 AJ23629916
–
AJ62011747
AJ22482617
–
EU145573*
–
Ixora coccinea L.
Kohautia caespitosa Schnizl.
Bremer et al. 42566B (UPS)
Kopsia fruticosa (Roxb.) A.DC.
Kraussia floribunda Harv.
RUB-Spermacoceae
21
Z68800
AM117324
8
APOCYNACEAE
IXO-Octotropideae
X91763
Z6885819
35
–
–
AJ235824
AM11732535 –
9
10
–
DQ1317466
AM295091
AM11736835
–
–
Lasianthus kilimandscharicus
K.Schum.
H. Lantz 119 (UPS)
RUB-Lasiantheae
AM11723735 AM11732735 EU145426*
EU145330*
DQ66214732
EU145366*
Lasianthus lanceolatus (Griseb.)
Urb.
Taylor 11719 (MO)
RUB-Lasiantheae
AM11723835 AF00406211
–
EU145331*
EU145554*
EU145367*
Lasianthus pedunculatus
E.A. Bruce
Andreasen 71 (UPS)
RUB-Lasiantheae
Z6880221
EU145504*
EU145427*
AJ2340032
EU145555*
EU145368*
Lasianthus strigosus Wight
Bremer & Bremer 3902 (UPS) RUB-Lasiantheae
AM11723935 EU145505*
EU145428*
–
EU145556*
EU145369*
Lerchea bracteata Valeton
Axelius 343 (S)
RUB-Ophiorrizeae
AJ2886102
EU145508*
EU145433*
AJ2339972
EU145561*
EU145374*
Luculia grandifolia Ghose
Bremer 2713 (S)
–Luculieae
X836481
AM90059360 AM11734635 AJ2339862
AJ3469293
AJ3468963
Luculia gratissima (Wall.) Sweet
Cult in Univ. Conn. Storres
870064 (no voucher)
–Luculieae
AM11724335 AJ4310364
AJ4309114
EU145344*
Luculia intermedia Hutch.
Howick et al. HOMC1524 (K) –Luculieae
–
EU145473*
EU145396*
–
EU145520*
–
Luculia pinceana Hook.
NN Thin et al. 3061 (AAU)
EU145447*
EU145472*
EU145395*
DQ1317496
AM1173713
–
Manostachya ternifolia
E.S. Martins
Bamps & Martins 4410 (UPS) RUB-Spermacoceae
AJ61621328
AM11732835 –
–
EU145572*
–
–
–
EU145568*
–
Margaritopsis nudiflora (Griseb.) Ekman 10248 (UPS)
K. Schum. (Syn. Margaritopsis
acuifolia)
Maschalocorymbus corymbosus
(Blume) Bremek.
Mitchella repens L.
Ridsdale 2471 (L)
Mitrasacmopsis quadrivalvis Jovet Kayombo et al. (UPS)
–Luculieae
AM11724735 AF00134011
RUB-Urophylleae
AJ2886112
AM90061160 –
–
EU145577*
EU145384*
RUB-Morindeae
Z6880521
AF00144111
–
–
AB10353554
AB103536
RUB-Spermacoceae
AJ61621428
AM11732935 EU145439*
EU145336*
EU145575*
EU145382*
AJ32007825
AJ23630016
AJ2340132
AF15261612
AY76284355
–
AJ2358289
DQ1316976
–
–
DQ1317546
EU145535*
AJ84685842
RUB-Morindeae
AJ318448
Mostuea brunonis Didr.
GELSEMIACEAE
L144048
Gillis 10838 (FTG)
Mycetia malayana (G. Don) Craib
Novotny et al. (2002)
1
IXO-Mussaendeae
X83652
RUB-Argostemmateae
Z6880621
CINCH-Naucleeae
RUB-Anthospermeae
1
X83653
X836541
25
–
16
EU145493*
AJ130836
AF00277111
–
AJ2340332
AF15262212
–
EU145410*
–
EU145320*
–
AJ3469583
AF15262312
AJ3468973
AF25792731
25
AJ320080
AF00274111
107
123
Nauclea orientalis (L.) L.
Coprosma granadensis Mutis ex
L.f. (syn. Nertera granadensis)
EU145308*
RUB-Psychotrieae
Morinda citrifolia L.
Mussaenda erythrophylla
Schumach. & Thonn.
AJ0119875
Deep divergences in the coffee family and the systematic position of Acranthera
Table 1 continued
108
123
Table 1 continued
Taxon
Voucher (of previously
unpublished sequences)
Classification
rbcL
rps16
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
Neurocalyx championii Benth. ex
Thwaites
Thor 601 (S)
RUB-Ophiorrizeae
EU145463*
EU145509*
EU145435*
–
EU145563*
EU145376*
Neurocalyx zeylanicus Hook.
B & K Bremer 937 (S)
RUB-Ophiorrizeae
Z6880721
AM90059460 EU145434*
35
31
AJ2339952
EU145562*
EU145375*
–
–
EU145543*
AF25793031
Normandia neocaledonica Hook.f. Munzinger 532 (MO)
RUB-Anthospermeae
AM117250
Oldenlandia corymbosa L.
Ophiorrhiza elmeri Merr.
RUB-Spermacoceae
RUB-Ophiorrizeae
X836551
EU145464*
AF33338120
EU145510*
AJ13083716
EU145436*
–
–
AF38153749
EU145564*
AY85405359
EU145378*
RUB-Ophiorrizeae
X836561
AF00406411
Ophiorrhiza mungos L.
Kjeldsen & Poulsen 233
(AAU)
Bremer 3301 (UPS)
2
Oreopolus glacialis (Poepp.)
Ricardi
RUB-Coussareeae
AJ288612
Paederia foetida L.
RUB-Paederieae
AF33237320
Palicourea crocea (Sw.) Schult
RUB-Psychotrieae
Palicourea guianensis Aubl.
RUB-Psychotrieae
Parapentas silvatica (K. Schum.)
Bremek.
AM117253
X83657
AJ13083816
–
DQ6621516
EU145377*
11
–
–
–
–
AF00406511
–
AJ2340062
AF15261912
–
AF147510
33
–
–
–
AF14932233
AF001345
11
AF004042
35
–
RUB-Knoxieae
AF257931
1
AM266849
37
–
–
AF152615
2
12
AM266937
37
AY63555456
AM26702337
–
AJ234021
EU145440*
EU145338*
AF10246743
EU145386*
Pauridiantha symplocoides
(S. Moore) Bremek.
Lantz 123 (UPS)
RUB-Urophylleae
AY53850215 AF00406811
Pauridiantha paucinervis (Hiern)
Bremek.
Bremer 3090 (UPS)
RUB-Urophylleae
Z6881121
AM90060060 AJ23630216
AJ2339982
EU145578*
EU145385*
Pentas lanceolata (Forssk.)
Deflers
RUB-Knoxieae
X836591
AM26687537 AJ23630416
X7647941
AM26696337
AB24727552
Pentodon pentandrus (Schumach.
& Thonn.) Vatke, Oesterr.
RUB-Spermacoceae
X836601
AF00361211
AJ2340242
–
–
–
Pouchetia baumanniana Büttner
(syn. Pouchetia gilletii)
Kiehn HBV sub RR-81-31
(WU)
IXO-Octotropideae
Z6885919
AM11733635 –
–
EU145541*
–
Praravinia suberosa (Merr.)
Bremek.
Sabah: Ridsdale no voucher
RUB-Urophylleae
AJ2886162
EU145514*
–
EU145579*
EU145387*
Pravinaria leucocarpa Bremek.
Beaman 7950 (S)
RUB-Urophylleae
AJ2886172
AM90061360 EU145441*
AJ2340012
EU145580*
EU145388*
X7648141
AY53846915
AF07203823
AF15261412
AF07199823
–
AF14940033
Psychotria kirkii Hiern
RUB-Psychotrieae
X83663
Psychotria pittieri Standl.
RUB-Psychotrieae
–
Psychotria poeppigiana Müll. Arg.
Pyrostria hystrix (Bremek.)
Bridson
RUB-Psychotrieae
Bremer 3791 (UPS)
1
AF410728
34
AF00274611
21
Z68818
AF002748
35
AM117262
Retiniphyllum pilosum (Spruce ex Wurdack & Adderley 43270
Benth.) Müll.Arg.
(S)
IXO-Retiniphylleae
AF33165420
Rhachicallis americana (Jacq.)
Hitchc.
CINCH-Rondeletieae
X836641
AM117338
35
AJ236307
16
–
–
–
AJ234018
2
47
AJ31511450
EU145418*
–
AJ620168
AF00407611
–
–
EU145536*
–
AF00407311
–
–
AF15274712
AY73030130
C. Rydin et al.
IXO-Vanguerieae
11
–
Taxon
Voucher (of previously
unpublished sequences)
Classification
rbcL
rps16
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
Rondeletia odorata Jacq.
Bremer & Andreasen 3504
(UPS)
CINCH-Rondeletieae
Y1185714
EU145490*
AJ2358459
EU145321*
AF15274112
AY73030730
RUB-Rubieae
X836661
–
DQ3591676
X7646541
–
DQ3588856
Rubia tinctorum L.
Sabicea aspera Aubl.
Andersson et al. 1941 (NY)
IXO-Sabiceeae
AY538508
15
AF004079
11
EU145416*
–
AY538475
15
Bremer et al. 4018-B18 (UPS) IXO-Sabiceeae
Bremer & al 4038-BB38 (UPS) RUB-Lasiantheae
EU145459* EU145494*
AM11726935 AF12927524
EU145415*
EU145429*
DQ131781
EU145332*
AJ847396
EU145557*
AJ84688342
EU145370*
Saldinia A. Rich. ex DC.
(specimen 2)
Kårehed et al. 257 (UPS)
RUB-Lasiantheae
EU145461*
EU145430*
EU145333*
EU145558*
EU145371*
Schismatoclada sp. Baker
Razafimandimbison &
Ravelonarivo 373 (MO)
Adam 20116 (UPS)
RUB-Danaideae
AM11727135 AM11734135 EU145425*
EU145329*
EU145553*
EU145365*
RUB-Schizocoleeae
AM11727235 EU14549861 –
EU14532361
EU14554661
EU145357*
RUB-Schradereae
Y1185914
AF00361711
AJ2340142
AF15261312
–
27
11
Schizocolea linderi (Hutch. &
Dalziel) Bremek.
Schradera sp K. Krause
–
42
AM409008
Sabicea diversifolia Pers.
Saldinia A. Rich. ex DC.
(specimen 1)
EU145506*
6
41
Sherardia arvensis L.
K. Andreasen 345 (SBT)
RUB-Rubieae
X81106
–
X76458
EU145571*
–
Sipanea biflora (L.f.) Cham. &
Schltdl.
Rova et al. 2005 (S)
IXO-Sipaneeae
AY53850915 AF00408511
EU145413*
DQ1317886
AF15267512
AY55511648
Sipanea hispida Benth. ex
Wernham
Irwin et al. 34756 (UPS)
IXO-Sipaneeae
EU145458*
EU145492*
EU145414*
EU145322*
AY55510748
AY55512248
IXO-Sipaneeae
–
AF24302230
–
Sipanea pratensis Aubl.
Spermacoce remota Lam.
RUB-Spermacoceae
Spigelia L.
Spiradiclis bifida Kurz
J. B. H. 55 (S)
21
Z68823
LOGANIACEAE
Y11863
14
AF004082
–
AF004093
11
–
AF15267712
AY55511548
AJ236309
16
–
–
–
AJ235840
9
–
–
AF17800451
RUB-Ophiorrizeae
EU145465*
EU145511*
EU145437*
–
EU145565*
EU145379*
Strychnos L.
LOGANIACEAE
L144108
AF00409411
AJ2358419
DQ1316916
AF10248443
–
Thecorchus wauensis (Schweinf.
ex Hiern) Bremek.
RUB-Spermacoceae
AM11728235 AM26690137 –
–
AM26698737
AM26707037
Theligonum cynocrambe L.
RUB-Theligoneae
X836681
Tricalysia cryptocalyx Baker
19
AF00408711
–
X8168040
AF15262112
–
11
–
–
AF15266912
AJ22482717
IXO-Coffeeae
Z68854
AF004088
Andersson & Nilsson 2304
(GB)
RUB-Lasiantheae
EU145462*
EU145507*
EU145431*
EU145334*
EU145559*
EU145372*
Trichostachys sp. Hook.f.
B. Sonké 1725 (UPS)
RUB-Lasiantheae
AJ2886262
AM90059560 EU145432*
DQ1317926
EU145560*
EU145373*
DQ1317936
EU145582*
–
AJ2340022
EU145581*
EU145389*
–
EU145542*
AJ22483917
60
Urophyllum arboreum (Reinw. ex Boeea 7887 (S)
Blume) Korth.
RUB-Urophylleae
–
AM900617
Urophyllum ellipticum (Wight)
Thwaites
Vangueria madagascariensis
J.F. Gmel.
Lundqvist 11085 (UPS)
RUB-Urophylleae
AJ2886272
AM90061960 –
Bremer 3077 (UPS)
IXO-Vanguerieae
X836701
–
–
AJ13084016
109
123
Trichostachys aurea Hiern
Deep divergences in the coffee family and the systematic position of Acranthera
Table 1 continued
110
123
Table 1 continued
Taxon
Voucher (of previously
unpublished sequences)
Classification
rbcL
rps16
ndhF
atpB-rbcL
spacer
trnT/F
nrITS
Virectaria major (K. Schum.)
Verdc.
Reekmans 10916 (UPS)
IXO-Sabiceeae
Y1186114
EU145495*
EU145417*
AJ2339892
EU145537*
EU145354*
Xanthophytum borneense
(Valeton) Axelius
Axelius 316 (S)
RUB-Ophiorrizeae
EU145466*
EU145513*
EU145438*
EU145335*
EU145567*
EU145381*
Xanthophytum capitellatum Ridl.
Ridsdale 2473 (L)
RUB-Ophiorrizeae
AJ2886282
EU145512*
–
AJ2339962
EU145566*
EU145380*
Total number of taxa in single
gene data sets
141
141
91
97
135
105
Total number of characters in
single gene data sets
1402
1602 ? 23
2243 ? 7
1098 ? 18
3219 ? 18
925 (677)**
Number of variable characters
527
1029
1172
605
1837
608 (386)**
Number of phylogenetically
informative characters
404
648
856
395
1145
504 (309)**
Evolutionary model employed
(AICc weights)
GTRIG
GTRG
GTRG
GTRG
GTRIG
GTRIG
Conflicts between Bayesian and
parsimony analyses
No
No
No
No
No
No
Conflicts between results
including/excluding indels
–
No
No
No
No
–
Notes. Classification: SUBFAMILY ABBREVIATION-Tribe. For outgroup taxa, only the FAMILY name is given. New classification in bold. Detailed information on methods and results is presented in
the text
* Previously unpublished sequence. ** Numbers within brackets represent values when parts of the nrITS alignment were removed. Published sequences: 1: Bremer et al. (1995). 2: Bremer and
Manen (2000). 3: Razafimandimbison and Bremer (2002). 4: Bremer et al. (2002). 5: Oxelman et al. (1999). 6: J-F Manen (GenBank unpublished). 7: Sennblad and Bremer (1996). 8: Olmstead
et al. (1993). 9: Backlund et al. (2000). 10: ME Endress et al. (GenBank unpublished). 11: Andersson and Rova (1999). 12: Rova et al. (2002). 13: Motley et al. (2005). 14: Bremer et al. (1998).
15: Andersson and Antonelli (2005). 16: Bremer et al. (1999). 17: Andreasen et al. (1999). 18: Andreasen and Bremer (2000). 19: Andreasen and Bremer (1996). 20: L Andersson (GenBank
unpublished). 21: Bremer (1996b). 22: Bremer (1996a). 23: Nepokroeff et al. (1999). 24: Piesschaert et al. (2000a). 25: Novotny et al. (2002). 26: Andersson (2001). 27: Manen and Natali
(1995). 28: Thulin and Bremer (2004). 29: Persson (2000). 30: JHE Rova (GenBank unpublished). 31: CL Anderson et al. (GenBank unpublished). 32: M Backlund (GenBank unpublished). 33:
L Andersson and C Taylor (GenBank unpublished). 34: Andersson (2002). 35: B Bremer (in prep.). 36: A Mouly (unpublished). 37: Kårehed and Bremer (2007). 38: Olmstead and Reeves
(1995). 39: XL Zhang et al. (GenBank unpublished). 40: Natali et al. (1995). 41: Manen et al. (1994). 42: Alejandro et al. (2005). 43: Struwe et al. (1998). 44: Yuan et al. (2003). 45: Gielly and
Taberlet (1996). 46: O Maurin et al. (GenBank unpublished). 47: Lantz and Bremer (2004). 48: Delprete and Cortes-B (2004). 49: Church (2003). 50: Lantz et al. (2002). 51: Gould and Jansen
(1999). 52: Nakamura et al. (2006). 53: P Ding et al. (GenBank unpublished). 54: J Yokoyama et al. (GenBank unpublished). 55: AD Proujansky and DL Stern (GenBank unpublished). 56: CW
Dick and E Bermingham (GenBank unpublished). 57: D Wolff and S Liede-Schumann (GenBank unpublished). 58: Church and Taylor (2005). 59: CI Yuan (GenBank unpublished). 60:
Smedmark et al. (2008). 61: Rydin et al. (2008)
C. Rydin et al.
Deep divergences in the coffee family and the systematic position of Acranthera
Table 2 Primers
Sequence 50 –30 /Reference
DNA region
Primer
names
rbcL
50 F, 30 R and Bremer et al. (2002)
427F
rbcL
Z895R
rps16
nrITS
F and 2R
Oxelman et al. (1997)
ITSForwRub CCTTATCATTTAGAGGAAGGAG
Zurawski, DNAX Research institute
nrITS
ITSRevRub
CCTCCGCTTATTGATATGC
nrITS
P17 and
26S-82R
Popp and Oxelman (2001)
nrITS
P25
Oxelman (1996)
ndhF
2F
Rydin et al. (2008)
ndhF
1000R
Rydin et al. (2008)
ndhF
720F
Rydin et al. (2008)
ndhF
1700R
Rydin et al. (2008)
ndhF
1320F
Rydin et al. (2008)
ndhF
2280R
Rydin et al. (2008)
atpB-rbcL
spacer
rbcL50 R
Rydin et al. (2008)
atpB-rbcL
spacer
atpB50 R
Rydin et al. (2008)
trnT-L-F
A1
Bremer et al. (2002)
trnT-L-F
940R
Rydin et al. (2008)
trnT-L-F
820F
Rydin et al. (2008)
trnT-L-F
IR
Bremer et al. (2002)
trnT-L-F
1250F
Rydin et al. (2008)
trnT-L-F
trnT-L-F
D
1880F
Taberlet et al. (1991)
Rydin et al. (2008)
trnT-L-F
2670R
Rydin et al. (2008)
ambiguous parts of nrITS (specified above). We used
Wilcoxon-signed rank tests implemented in VassarStats
(Lowry 2008) to test for significant changes in posterior
probabilities and bootstrap estimates between analysis
including or excluding nrITS.
Parsimony analyses were performed in Paup* version
4.0b10 for Unix (Swofford 1998), for single gene data sets,
as well as for combined data sets including and excluding
Table 3 Results of selected
combined analyses
111
nrITS. Most parsimonious trees were calculated using the
heuristic search option, 500 random sequence additions,
tree bisection reconnection branch swapping. Support
values were obtained by using bootstrap in Paup*, performing 1,000 bootstrap replicates, each with 10 random
sequence additions with settings as before. A majority rule
consensus tree was produced from the resulting trees,
in which nodes with a bootstrap support \50% were
collapsed.
Pollen morphology
Anthers with in situ pollen of Acranthera tomentosa R.Br.
ex Hook.f., voucher: Vidal 5001 (P), were mounted on
cleaned aluminium stubs and initially investigated under a
stereomicroscope. For scanning electron microscopy
(SEM), the material was coated with gold for 90 s in a
sputter coater, and examined with a Hitachi Field Emission
scanning electron microscope at 5 kV.
Results
Data
The aligned six-gene data set included 149 terminals and
10,555 characters, from which 1,402 derived from rbcL,
1,602 from rps16, 2,243 from ndhF, 1,098 from atpB–rbcL
spacer, 3,219 from trnT–L–F: 3,219, 925 from nrITS and
66 from indels (see also Table 1). The nrITS alignment
with potentially ambiguous parts removed contained 677
characters. The number of variable and informative characters, number of supported nodes and average support
values are given for single gene analyses in Table 1 and for
combined analyses in Table 3.
Model choice
For each single gene analysis, the best performing model
according to the corrected Akaike information criterion
5 regions ? 6 regions ? 6 regions ? indels
6 regions
indels
indels
(parts of nrITS removed) (no indels)
Number of characters in matrix
9,630
10,555
10,307
10,489
Number of variable characters
5,228
5,778
5,614
5,712
Number of informative characters
3,449
3,952
3,757
3,886
Number of supported nodes (bootstrap) 120
129
128
128
Number of supported nodes (Bayesian) 133
136
–
133
Average support (bootstrap)
90.84
92.15
89.56
90.60
Average support (Bayesian)
96.60
97.15
–
96.66
123
112
C. Rydin et al.
100 (100)
95 (82) 100 (100)
87 (71) 100 (100)
53 (58)
100 (100)
57 (70)
100 (100)
100 (100)
100 (100)
100 (100)
98(100)
91 (91)
100 (100)
100 (100)
100 (100)
88 (90)
93 (---)
63 (---)
78(85)
100 (100) --- (---)
100 (100)
100 (100)
100 (100)
72(67)
79 (78)
100 (100)
100 (100)
100 (100)
91 (90)
100 (---)
96 (---)
100 (100)
100 (100)
100 (---)
--- (---)
100 (100)
100 (84)
100 (---)
81 (55) 100 (99) 100 (---)
85 (---)
99 (89)
100 (100)
100 (100) 100 (100)
100 (---) 100 (100)
57 (---) ( --- )(---)
66 (---)
96(---)
100 (100)
85 (---)
100 (100)
92 (86)
100 (100)
Cinchonoideae
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
87 (60) 100 (100) 67 (82)
76 (81)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (98)
100 (100)
98 (96)
95(58)
74 (---)
100 (100)
100 (100)
99 (83)
92 (---)
59 (---)
100 (100) ---(---)
98 (97)
100 (100)
100 (100)
100 (100)
94 (72)
100 (100)
100 (100)
Ixoroideae
100 (100)
82 (---)
100 (100)
100 (100)
96 (92)
90 (56)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100) 100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
82 (82)
100 (100)
100 (100)
72 (88)
55 (54)
100 (100)
100 (100)
Alstonia
Kopsia
Anthocleista
Gentiana
Gelsemium
Mostuea
Strychnos
Spigelia
Luculia grandifolia
Luculia gratissima
Luculia pinceana
Luculia intermedia
Acranthera grandiflora
Acranthera frutescens
Acranthera sp. 1
Acranthera atropella
Acranthera sp. 2
Acranthera siamensis
Acranthera siamensis?
Coptosapelta montana
Coptosapelta flavescens 2
Coptosapelta flavescens 3
Coptosapelta diffusa 1
Coptosapelta diffusa 2
Coptosapelta flavescens 1
Coptosapelta tomentosa 1
Coptosapelta tomentosa 2
Catesbaea spinosa
Cubanola domingensis
Chiococca alba
Cinchona pubescens
Hillia triflora
Cephalanthus occidentalis
Nauclea orientalis
Hymenodictyon floribundum
Guettarda uruguensis
Rhachicallis americana
Rondeletia odorata
Alberta magna
Bertiera guianensis
Coffea arabica
Tricalysia cryptocalyx
Diplospora polysperma
Cremaspora triflora
Feretia aeruginescens
Fernelia buxifolia
Kraussia floribunda
Pouchetia baumanniana
Aidia micrantha
Ixora coccinea
Pyrostria hystrix
Vangueria madagascariensis
Retiniphyllum pilosum
Mussaenda erythrophylla
Sabicea diversifolia
Sabicea aspera
Virectaria major
Emmenopterys henryi
Condaminea corymbosa
Calycophyllum candidissimum
Ferdinandusa speciosa
Sipanea biflora
Sipanea hispida
Sipanea pratensis
Outgroup
Luculieae
Coptosapelteae
Chiococceae
Cinchoneae
Hillieae
Naucleeae
Hymenodictyeae
Guettardeae
Rondeletieae
Alberteae
Bertiereae
Coffeeae
Cremasporeae
Octotropideae
Gardenieae
Ixoreae
Vanguerieae
Retiniphylleae
Mussaendeae
Sabiceeae
Condamineeae
Sipaneeae
Rubioideae
Fig. 1 Relationships within the tribes Luculieae and Coptosapelteae;
and the subfamilies Cinchonoideae and Ixoroideae, estimated using
Bayesian inference of phylogeny based on molecular data from
chloroplast regions rbcL, rps16 intron, ndhF, atpB–rbcL spacer,
trnT–L–F, the nuclear ribosomal ITS and indels. Posterior probabilities are given above branches, bootstrap values (under parsimony)
below. Support values from the analyses of chloroplast data (excluding nrITS) are given (in brackets)
(AICc, Akaike 1973) was selected. AICc is appropriate
when the ratio between sample size and number of
parameters is small (n/K \ 40, Burnham and Anderson
2003, p. 66), but also for higher ratios because AICc will
then converge to AIC (Posada and Buckley 2004).
Empirically, the three criteria indicated the same best
performing model for all matrices. For the rbcL, trnT–L–F
and nrITS data, the general time reversible model (Tavare
123
Deep divergences in the coffee family and the systematic position of Acranthera
100 (100)
100 (99)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
52 (97)
96(96)
--- (---)
87 (98)
100 (100)
100 (100) (57)
69 (78) 100 (100)100 (100) 10063(100)
--- (76)
98 (97)
100 (100)
--- (---)
100 (100)
100 (100)
80(---) 100 (100)
100 (100)
94 (---) 100 (100)
100 (98)
100 (100)
100 (100)
100 (100)
100 (100) --- (95)
100 (100)
98 (99)
100 (100)
Spermacoceae alliance
100 (100)
100 (100)
100 (98)
--- (---)
75 (54)
63 (51)
100 (100)
97 (100)
98 (100)
52 (75)
100 (100)
100 (100) 60(---)
--- (---)
69(50)
--- (---)
100(100)
100 (100)
100 (100)
100 (100)
100 (100)
99(99) 82 (80)
100 (100) 66 (69) 100 (100)
98 (97)
100 (100) 79 (84)
100 (100)
96 (87) 54 (57)
88 (64)
100 (100) --- (---)
82 (84)
100 (100)
99 (96)
100 (100)
100 (99)
100 (100)
100 (100)
97 (98)
93 (98) 97 (96)
85 (86)
100 (---)
100 (100)
100 (100) 100 (---)
90 (92)
97 (99)
100
(100)
100 (100)
--- (80)
94 (95) 100 (100)
100 (100)
97 (98)
100 (100)
85 (59)
100 (100)
--- (---)
100 (100)
100 (100)
100 (100) 100 (100)
(100)
89 (96) 100 (100) 100
100 (100)
88 (84) 100 (100)
100 (100)
100 (100)
100 (100) 100 (100)
98 (100)
99 (98)
100 (100) 56 (---)
100 (100) 70 (66)
100 (100)
100 (100)
100 (100) 100 (100)
100 (100) 100 (100)
100 (100)
100 (100)
100 (79)
100
(100)
100
(100)
100 (100)
100 (100)
100
(100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
Psychotrieae alliance
100 (100)
99 (99)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
95 (99)
100 (100)
100 (100)
100 (---)
82 (66)
100 (100)
100 (100)
100 (100)
100 (100) 98 (90)
100 (100)
100 (100)
90 (93) 71 (96)
53 (78) 100 (100)
100 (100)
100 (100)
100 (100)
Rubioideae
Coprpsma granadensis
Normandia neocaledonica
Anthospermum herbaceum
Mycetia malayana
Argostemma hookeri
Paederia foetida
Didymaea alsinoides
Rubia tinctorum
Sherardia arvensis
Galium album
Theligonum cynocrambe
Dunnia sinensis 1
Dunnia sinensis 2
Dunnia sinensis 3
Dunnia sinensis 4
Dunnia sinensis 5
Danais xanthorrhoea
Schismatoclada sp
Batopedina pulvinellata
Pentas lanceolata
Dirichletia glaucescens
Parapentas silvatica
Hedyotis fruticosa
Manostachya ternifolia
Kohautia caespitosa
Bouvardia ternifolia
Spermacoce remota
Oldenlandia corymbosa
Thecorchus wauensis
Unknown Rubiaceae
Arcytophyllum aristatum
Houstonia caerulea
Dibrachionostylus kaessneri
Mitrasacmopsis quadrivalvis
Dentella repens
Pentodon pentandrus
Schizocolea linderi
Morinda citrifolia
Gynochthodes coriacea
Damnacanthus indicus
Mitchella repens
Schradera subandina
Palicourea guianensi
s
Palicourea crocea
Psychotria poeppigiana
Psychotria pittieri
Margaritopsis nudiflorav
Geophila obvallata
Psychotria kirkii
Cremocarpon lantzii
Hydnophytum formicarum
Coccocypselum condalia
Coccocypselum hirsutum
Declieuxia cordigera
Declieuxia fruticosa
Cruckshanksia hymenodon
Oreopolus glacialis
Coussarea macrophylla
Coussarea hydrangeifolia
Faramea multiflora
Lasianthus kilimandscharicus
Lasianthus pedunculatus
Lasianthus lanceolatus
Lasianthus strigosus
Saldinia sp. 1
Saldinia sp. 2
Trichostachys aurea
Trichostachys sp.
Lerchea bracteata
Ophiorrhiza mungos
Ophiorrhiza elmeri
Spiradiclis bifida
Xanthophytum capitellatum
Xanthophytum borneense
Neurocalyx zeylanicus
Neurocalyx championii
Amphidasya ambigua
Maschalocorymbus corymbosus
Praravinia suberosa
Pravinaria leucocarpa
Urophyllum arboreum
Urophyllum ellipticum
Pauridiantha paucinervis
Pauridiantha symplocoides
Colletoecema dewevrei
113
Anthospermeae
Argostemmateae
Paederieae
Rubieae
Theligoneae
Dunnieae
Danaideae
Knoxieae
Spermacoceae
Schizocoleeae
Morindeae
Mitchelleae
Schradereae
Psychotrieae s.l.
Coussareeae
Lasiantheae
Ophiorrhizeae
Urophylleae
Colletoecemateae
Fig. 2 Relationships within subfamily Rubioideae, estimated using
Bayesian inference of phylogeny based on molecular data from
chloroplast regions rbcL, rps16 intron, ndhF, atpB–rbcL spacer,
trnT–L–F, the nuclear ribosomal ITS and indels. Posterior
probabilities are given above branches, bootstrap values (under
parsimony) below. Support values from the analyses of chloroplast
data (excluding nrITS) are given (in brackets)
1986) with gamma distributed rates (Yang 1993) and a
proportion of invariable sites was selected (GTR ? I ? C).
For the rps16, ndhF and the atpB-rbcL spacer, GTR ? C
was selected (Table 1). For combined analyses with less
than seven partitions, GTR ? C was selected for the
chloroplast partition.
123
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C. Rydin et al.
Analyses
Table 4 Test for significance of differences in support values, when
including/excluding nrITS
Combined data set
Including/excluding nrITS
z
P (twotailed)
Bayesian posterior probabilities
(Rubiaceae)
z = 0.98
P = 0.3271
As described in ‘‘Materials and methods’’, the combined
data set was analysed several times, partitioning the data
set in different ways. These analyses resulted in nearly
identical topologies, but with slight differences in resolution and support values. We observed no supported
(i.e. C50% posterior probability and/or bootstrap support)
conflicts between results obtained from the different combined analyses. Figures 1, 2 show the results from the
Bayesian analysis including information from indels (two
data partitions: nucleotide data and indels). Bootstrap values of 50% or more are plotted on the Bayesian tree. We
have further indicated (within brackets) support values
from the 5-cp gene analysis (excluding nrITS).
Usefulness of nrITS for addressing deep divergences
in Rubiaceae
Including nrITS generally increased resolution and support
values over the entire phylogeny (Figs. 1, 2; Table 3).
Some nodes received a lower support when nrITS
was added, but overall resolution and average support
(arithmetic mean) increased. The phylogeny based on
rbcL, rps16, ndhF, the atpB–rbcL spacer and trnT–L–F
(excluding nrITS) had 120 supported nodes with an average bootstrap value of 90.84%. The tree also based on
nrITS data had 129 supported nodes with an average
bootstrap support of 92.15%. For Bayesian analyses, the
analysis excluding nrITS had 133 supported nodes with an
average posterior probability of 96.60%. Including nrITS
yielded 136 supported nodes with an average posterior
probability of 97.15%. However, the increase in mean
support values was not statistically significant (Table 4),
neither in Bayesian analyses (z = 0.98, P = 0.327), nor in
bootstrap analyses (z = 0.92, P = 0.358). For subfamily
Rubioideae, mean bootstrap support was slightly lowered
when including nrITS, but the difference was not significant (z = -0.46, P = 0.6455). In Bayesian analyses,
support values increased also in Rubioideae when
including nrITS, but again not significantly (z = 0.16,
P = 0.8729).
The topology from the analysis of six genes, excluding
potentially ambiguous sites in nrITS, was basically the
same as for the complete six-gene topology but support
values generally decreased and some resolution was lost
(Table 3). The sister relationship between Luculia and the
Acranthera–Coptosapelta clade was for example collapsed
in this tree, as was the case in the 5-cp analysis, excluding
nrITS altogether (Fig. 1).
123
Bayesian posterior probabilities (clade Ab) –a
b
a
Bayesian posterior probabilities (clade B ) –
b
–a
–a
Bayesian posterior probabilities (clade C ) z = 0.16
P = 0.8729
Bootstrap values (Rubiaceae)
z = 0.92
P = 0.3576
Bootstrap values (clade A)
–a
–a
Bootstrap values (clade B)
z = 1.55
P = 0.1211
Bootstrap values (clade C)
z = -0.46 P = 0.6455
Wilcoxon signed-rank test
a
ns/r too small
Clade A: Luculia–Coptosapelta–Acranthera; clade B: Cinchonoideae-Ixoroideae; clade C: Rubioideae
b
Single gene analyses
We found no major conflicts between single gene data sets
and no conflicts within each region (between parsimony
and Bayesian analyses, or when including or excluding gap
information, see also Table 1). The position of a few taxa
varied between single gene data sets and supported deviations are presented below.
Phylogeny—the combined data set
Deep divergences and the Luculia–Acranthera–
Coptosapelta clade
All ingroup taxa were resolved in three (or four) major
clades (Figs. 1, 2). 1: The Luculia–Acranthera–Coptosapelta clade (which collapsed in the 5-cp analysis into one
Luculia clade and one Acranthera–Coptosapelta clade); 2:
the Cinchonoideae–Ixoroideae clade; 3: the Rubioideae
clade. Support was very high for the latter two groups
(support values are presented as follows [Bayesian posterior probability including nrITS (posterior probability
excluding nrITS)/bootstrap support including nrITS
(bootstrap support excluding nrITS)]: Cinchonoideae–
Ixoroideae [100 (100)/100 (98)] and Rubioideae [100
(100)/100 (100)]. Luculia, Acranthera and Coptosapelta
fell outside these groups. Acranthera and Coptosapelta
were sister groups in all analyses [100 (100)/100 (100)], a
result which to our knowledge has not been presented
before. Luculia was sister to the Acranthera–Coptosapelta
clade with relatively high Bayesian posterior probability,
but low bootstrap support and only recovered when information from the entire nrITS was included [93 (–)/63 (–)].
Deep divergences in the coffee family and the systematic position of Acranthera
All currently recognised species of Luculia were included in this study and we show that the genus is
monophyletic [100 (100)/100 (100)]. Our results also
support the monophyly of Acranthera [100 (100)/100
(100)] and Coptosapelta [100 (100)/100 (100)].
Results within the Cinchonoideae–Ixoroideae clade
Support values for Cinchonoideae and Ixoroideae (Fig. 1)
were high [100 (100)/100 (100)]. Within Cinchonoideae,
Rondeletieae–Guettardeae [100 (100)/100 (100)] was sister
to a large clade comprising Hymenodictyeae, Naucleeae,
Hillieae, Cinchoneae and Chiococceae [96 (–)/85 (–)].
Hymenodictyeae and Naucleeae formed a clade [100 (100)/
100 (100)]. Hillieae was sister to Cinchoneae and Chiococceae [100 (–)/– (–)].
Within Ixoroideae, Sipaneeae and Condamineeae were
sister groups [99 (83)/92 (–)], and this clade was sister to
remaining Ixoroideae [96 (92)/90 (56)]. Sabiceeae and
Mussaendeae [100 (100)/94 (72)] comprise the next
diverging clade, followed by Retiniphyllum. The position
of Retiniphyllum was strongly supported. Within remaining
Ixoroideae, Vanguerieae and Ixoreae [100 (100)/100 (100)]
were sister to a clade comprising Alberta, Coffeeae,
Bertiereae, Cremasporeae, Octotropideae and Aidia
[100 (100)/100 (100)], within which Alberta was sister to
the two sister clades [100 (100)/100 (100)]: Coffeeae–
Bertiereae [100 (100)/87 (60)] and Cremasporeae–
Octotropideae–Aidia [95 (58)/74 (–)]. Within the latter,
Aidia was sister to a Cremasporeae–Octotropideae clade
[100 (100)/82 (–)].
Results within Rubioideae
Subfamily Rubioideae (Fig. 2) was well supported [100
(100)/100 (100)]. Colletoecema dewevrei was sister to
remaining Rubioideae with high support [100 (100)/95
(99)]. The next diverging clade consisted of Urophylleae
[100 (100)/100 (100)] and Ophiorrhizeae [100 (100)/100
(100)], which grouped together with relatively high support
[100 (–)/82 (66)]. Lasiantheae [100 (100)/100 (100)] was
the next diverging group, followed by Coussareeae [100
(100)/100 (100)], which was sister group to the Psychotrieae and Spermacoceae alliances [98 (100)/52 (75)].
The Psychotrieae alliance [100 (100)/100 (100)] was here
represented by 15 species. Schizocolea linderi was highly
supported as sister to a clade comprising the remaining
sampled taxa [100 (100/90 (92)]. The remaining species
comprised two sister groups: 1) Mitchelleae–Schradereae
[93 (98)/85 (86)], sister to Morindeae [100 (100)/100 (99)],
and 2) Psychotrieae s.l. [100 (100)/100 (100)].
The Spermacoceae alliance [100 (100)/100 (100)]
comprised two major clades: The first was here represented
115
by Anthospermeae, Argostemmateae, Paederieae, Rubieae,
Theligoneae and Dunnieae [100 (100)/52 (97)]. Amongst
these, Anthospermeae [100 (100)/100 (100)] was the earliest diverging group, followed by Argostemmateae [100
(100)/100 (100)]. The next diverging clade [– (76)/ –(–)]
comprised Dunnieae [100 (100)/100 (100)] and its sister
clade [100 (100)/69 (78)], which consisted of Paederieae
and Rubieae–Theligoneae [100 (100)/100 (100)]. Within
the second major clade of the Spermacoceae alliance [100
(98)/ –(–)], Danaideae [100 (100)/100 (100)] was sister to
Knoxieae–Spermacoceae [100 (100)/97(100)].
Phylogeny—single gene data sets
Generally, single gene analyses produced the same topologies as those obtained from the combined data set. There
are some minor deviations and we arbitrarily decided that
differences with a Bayesian posterior probability higher
than 85%, and/or a bootstrap support higher than 50% can
be considered ‘‘supported’’. Such differences are presented
here (posterior probability/bootstrap index).
rbcL
The results from the Bayesian analysis of the rbcL data
resolved Ophiorrhizeae and Urophylleae as a basal grade
(instead of a clade) within Rubioideae (95/– for Rubioideae
except Ophiorrhizeae). The result was not supported in the
bootstrap analysis of the rbcL data.
rps16
The analyses of the rps16 data resolved Luculia, Acranthera and Coptosapelta as sister to the Cinchonoideae–
Ixoroideae clade (93/76).
ndhF
In the ndhF tree, the Acranthera–Coptosapelta clade was
sister to remaining Rubiaceae including Luculia (98/85).
Colletoecema was sister to Lasiantheae (90/–). This relationship was not supported in bootstrap analyses.
Sipaneeae and Condamineeae formed a basal grade (not a
clade) within Ixoroideae. Support for Condamineeae and
remaining Ixoroideae was low (55/80).
trnT–L–F
In analyses based on the trnT–L–F data, the Acranthera–
Coptosapelta clade was sister to Rubioideae (88/80). There
were some differences amongst major clades in the Spermacoceae alliance, regarding the positions of Danaideae,
Anthospermeae and Argostemmateae. The differences had
123
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C. Rydin et al.
The nrITS data resolved Colletoecema as sister to the
Urophylleae–Ophiorrhizeae clade (82/76). Coussareeae
grouped together with a collapsed Anthospermeae (95/–).
This relationship was not supported in the bootstrap
analysis.
protrusion. The sexine is (micro)reticulate-perforate but
differs probably between mesocolpial and apocolpial
areas. Structures tentatively interpreted as aborted grains
(ovoid, about 3 lm long, roughly undulating-palliate
surface and apertures, not shown), were numerously
present amongst the grains. Note: this SEM study represents a preliminary overview of characters found in
grains (not acetolysed) from one specimen. Further
studies are needed to provide more details and detect
potential inter and intraspecific variation in Acranthera
pollen.
Pollen morphology
Taxonomic implications
Because our results strongly support Acranthera as sister
to Coptosapelta, which has unique pollen morphology
(Verellen et al. 2004), we made a preliminary SEM study
of Acranthera pollen. Acranthera pollen (Fig. 3) is triangular (rarely quadrangular) in shape and spheroidal to
subspheroidal, with a polar axis of about 17 lm and
equatorial diameter of 18–22 lm. They have three (rarely
four) apertures positioned at the angles. The apertures are
of a compound, colporate type. The ectoaperture is a
short colpus (6–8 lm long), with acute to obtuse endings.
The mesoaperture is a pore with a diameter of about
3–4 lm. Each mesoaperture is covered by an apertural
Based on the results, we describe four new tribes and one
new tribal circumscription. Our decisions are based on the
principles of classification outlined in Backlund and Bremer (1998).
Acranthera is strongly supported as sister to Coptosapelta and we have included Acranthera in the tribe
Coptosapelteae.
Considering the persisting difficulties to find support for
a close relationship between Luculia and other species of
Rubiaceae, we have chosen to describe the new monogeneric tribe Luculieae. Luculieae and Coptosapelteae are
clearly excluded from the three subfamilies Ixoroideae,
a Bayesian posterior probability of 80–90% but were not
present in the bootstrap tree. These results are further
investigated elsewhere.
nrITS
Fig. 3 Pollen grains of
Acranthera tomentosa (SEM): a
Polar view. Acranthera pollen is
generally triangular in shape,
spheroidal to subspheroidal and
about 18–22 lm in equatorial
diameter. The sexine is
(micro)reticulate-perforate. The
grains have three apertures
positioned at the angles.
b Equatorial view. c The
apertures are of a compound,
colporate type; the ectoaperture
is a short colpus and the
mesoaperture is a pore. Each
mesoaperture is covered by an
apertural protrusion. d Polar
view. Acranthera grains are
rarely quadrangular with four
apertures positioned at the
angles. Scale bars 5 lm
123
Deep divergences in the coffee family and the systematic position of Acranthera
Cinchonoideae and Rubioideae, but we do not propose a
new subfamily for the Luculia–Coptosapelta–Acranthera
clade at this point. The clade is relatively well supported
(93%) in the Bayesian analysis of the six-gene data set, but
poorly supported in bootstrap analysis (63%), and collapsed in five-gene data sets. Further studies are needed
to confirm the monophyly of the Luculia–Acranthera–
Coptosapelta clade.
Three genera, Colletoecema, Schizocolea and Dunnia,
are lone sister lineages of large clades comprising several
well-defined tribes. They cannot be implemented in any of
the existing tribes and we have therefore described the new
monogeneric tribes Colletoecemateae, Schizocoleeae and
Dunnieae (see below).
Discussion
In order to address deep divergences in Rubiaceae, we
sampled a large data set comprising 149 terminals and
nearly 11,000 characters. The project has thus had potential
to address a number of previously unresolved relationships
and conflicting results throughout the family. Morphology
and character evolution are discussed but obvious morphological support for major groups defined by molecular
data may be difficult to find.
The usefulness of nrITS
Nuclear ribosomal ITS has previously been used for
resolving higher-level relationships within Rubiaceae (e.g.
Andreasen et al. 1999) but not for addressing the phylogeny of the entire family. A comparison of the topologies
from analyses including and excluding nrITS shows that
when nrITS is included, resolution and/or support increase
for relationships within several groups of interest here, for
example, the sister relationships between Urophylleae and
Ophiorrhizeae, between Sipaneeae and Condamineeae and
between Luculia and the Coptosapelta–Acranthera clade
(Figs. 1, 2).
There are also nodes (for example in the Spermacoceae
alliance), for which support values decrease when nrITS is
included and we conducted a bootstrap analysis on the
combined six-gene data set, excluding two short regions of
nrITS where homology assessments were difficult and
potentially ambiguous. The resulting topology was nearly
identical to that obtained from the complete six-gene data
set, but slightly less well resolved and with a distinctly
lower average support value (Table 3). In the present
study, nrITS thus provided structured information, which
resulted in increased resolution. Nuclear ITS also contributed to an increase in average support, however, many
nodes were well-supported also without information from
117
nrITS and the increase in support values was not statistically significant.
New insights into evolutionary relationships—
Acranthera
The sister relationship between Acranthera and Coptosapelta is very well supported in all combined and single
gene analyses except in the analysis of nrITS, where the
node is present but less well supported (94/–). Our results
further support the monophyly of the two genera. To our
knowledge, these results have not been presented before.
Although the Acranthera–Coptosapelta clade is well
supported by molecular data, we find no unambiguous
morphological support for the relationship. Bremekamp
(1947, p. 273) discussed a potential synapomorphy for
Acranthera and Coptosapelta: the style functioning as a
temporary depository for pollen, a ‘‘receptaculum pollinis’’. However, Puff et al. (1995) considered such a
structure in Acranthera a misconception and they consequently refuted this synapomorphy for Coptosapelta and
Acranthera. Further, even though Bremekamp (1947)
suggested secondary pollen presentation as a potential
synapomorphy for Acranthera and Coptosapelta, he argued
that the united apical connective appendage in Acranthera
is a feature unique within Rubiaceae and similar to the
morphology of stamens in Apocynaceae. Puff et al. (1995)
also considered the ‘‘anther–style and stigma complex’’ of
Acranthera unique within Rubiaceae, in structure as well as
function.
Pollen grains of Coptosapelta possess several features
unique within Rubiaceae (Verellen et al. 2004). They are
pororate and may have up to 10 apertures (even if 3–4
apertures are most common), they lack columellae and they
have ‘‘droplets’’ on the inner nexine (Verellen et al. 2004).
Acranthera pollen has so far not been thoroughly documented (but see Mathew and Philip 1983) and in order to
get an indication on whether Acranthera pollen shares
some of the features of Coptosapelta grains, we performed
a preliminary SEM study of the outer surface of the grains
and the nature of the apertures (Fig. 3).
Several characters of Acranthera pollen are common in
Rubiaceae and probably plesiomorphic. Acranthera grains
are not pororate (like Coptosapelta grains) but colporate,
which is considered the plesiomorphic condition in the
family (Dessein et al. 2005). The size of Acranthera grains
(18–22 lm in equatorial diameter) fits within the 20–
40 lm, which is most common in Rubiaceae (Dessein et al.
2005). The triangular (rarely quadrangular) shape is more
unusual but occurs according to Dessein et al. (2005) for
example in Tapiphyllum Robyns (i.e. Vangueria Juss.) and
Psydrax Gaertn. (Vanguerieae, Ixoroideae). Apertural
protrusions (papillae-forming onci), pollen buds and
123
118
structures that cover the aperture (opercula) have been
reported for several genera of Rubiaceae, but to our
knowledge, not for Coptosapelta.
There are some potential similarities between Acranthera and Coptosapelta pollen. The short ectocolpi of
Acranthera could perhaps be compared with the ectopores
of Coptosapelta and the microreticulate to perforate sexine
in Acranthera is similar to that described for some species
of Coptosapelta (Verellen 2002). However, pollen characters in Acranthera need to be further studied (e.g. the
presence or absence of columellae, ‘‘droplets’’ on the inner
nexine, the nature of the apertural protrusions) before any
hypotheses on synapomorphies can be put forward.
The enigmatic Luculia
Our study included all four currently recognised species of
Luculia (Govaerts et al. 2006) and we show that the genus
is monophyletic, but its relationship to other species of
Rubiaceae remains uncertain. The clade comprising Luculia, Acranthera and Coptosapelta is here only supported
in some of the single gene analyses (atpB-rbcL spacer and
nrITS) and in combined analyses including nrITS. However, no analysis resulted in a well-supported alternative
position for Luculia. Further, there is biogeographical
support for a relationship between these three South East
Asian genera and a relationship between Luculia and
Coptosapelta has been indicated in other recent studies
(Robbrecht and Manen 2006).
The Luculia–Acranthera–Coptosapelta clade is equally
puzzling from a morphological perspective as is the
Acranthera–Coptosapelta clade. Korthals (1851) very
briefly mentioned some similarities between Luculia and
Coptosapelta regarding the form of the seed, but he did not
specify this further. Bremekamp (1947, p. 261) considered
corolla aestivation, insertion of the stamens in the corolla
tube and many-seeded fruits important regarding the systematic position of Acranthera, but these characters
provide no support for the Luculia–Acranthera–Coptosapelta clade. Corolla aestivation is imbricate in Luculia
(Bremer and Struwe 1992), valvate in Acranthera
(Bremekamp 1947) and contorted in Coptosapelta
(Andersson and Persson 1991). Filaments are inserted at
the base of the corolla tube in Acranthera (Bremekamp
1947), but at about one-third from the mouth of the corolla
tube in Coptosapelta and Luculia (Andersson and Persson
1991). All three genera have many-seeded fruits (Sweet
1826; Korthals 1851; Bremekamp 1947), but this character
is common in Rubiaceae and probably plesiomorphic.
Pollen characters also show little resemblance between
the three genera. Luculia grains are small to medium-sized,
22–24 lm in polar axis (Murray 1990), spheroidal, 3(–4)colporate and with a reticulate tectum (Dessein et al. 2005).
123
C. Rydin et al.
These character states probably represent primitive states
within the family (Dessein et al. 2005) so even though
grains of Coptosapelta are (oblate)spheoidal (Verellen
et al. 2004), and Acranthera grains are (tri)colporate (the
present study), these respective similarities with Luculia
grains are likely plesiomorphic. The more specialised
respective features of Coptosapelta and Acranthera pollen,
e.g. the pororate pollen of Coptosapelta and the triangular
shape of Acranthera grains, are not present in Luculia.
Early divergences within the family
Despite that we have used a relatively extensive sampling
of taxa and characters in this study, the major clades of the
family form a basal trichotomy: (1) the Luculia–Acranthera–Coptosapelta clade, (2) a clade consisting of the
subfamilies Cinchonoideae and Ixoroideae, (3) subfamily
Rubioideae (Figs. 1, 2).
Robbrecht and Manen (2006) argued, based on parsimony analyses of 15 selected species and eight gene
regions, that Luculia and Coptosapelta (Acranthera was
not investigated) are ‘‘basal to the rest of Cinchonoideae’’
(i.e. sister to the Cinchonoideae–Ixoroideae clade). However, this conclusion is not supported by their results. Their
combined analysis had no support for the position of these
genera (Robbrecht and Manen 2006, Fig. 2) and the super
tree analysis placed Luculia and Coptosapelta as sister to
the rest of the family, not sister to the Cinchonoideae–
Ixoroideae clade (Robbrecht and Manen 2006, Fig. 4a).
Results from super tree analyses are difficult to evaluate;
trees from the literature often contain some poorly supported nodes, which consequently may decrease accuracy
of the super tree. Further, some information in the original
data sets is lost, because the character information is simplified into a phylogeny (de Queiroz and Gatesy 2007).
When analysing a combined data set, it is possible to get
increased support for relationships that are not supported,
perhaps not even present, in the single gene analyses (see
e.g. Kluge 1989; Olmstead and Sweere 1994). This has,
however, not been the case regarding basal relationships in
Rubiaceae. Different gene regions produce contradicting
(poorly supported) results and the combined analyses are
unresolved (the present study and Robbrecht and Manen
2006).
Ixoroideae
Sipaneeae and Condamineeae form a strongly supported
clade, which is sister to the remaining Ixoroideae.
Sabiceeae and Mussaendeae are sisters (see also Alejandro
et al. 2005) and comprise the next diverging clade, followed by Retiniphylleae. Two additional well-supported
relationships within Ixoroideae have not been presented
Deep divergences in the coffee family and the systematic position of Acranthera
119
In our study, Rondeletieae and Guettardeae form a clade,
sister to the remaining Cinchonoideae. The result is well
supported but differs from that reported in Andersson and
Antonelli (2005), where Naucleeae and Hymenodictyeae
constituted the sister clade to the remaining Cinchonoideae. The sister-group relationship between Naucleeae and
Hymenodictyeae, previously shown by Razafimandimbison
and Bremer (2001) and later endorsed by Andersson and
Antonelli (2005), is further supported by our analyses, as
well as by pollen morphology (Verellen et al. 2007).
However, an extended sampling in Cinchonoideae is needed to further address the relationships and evolution of the
group (see Manns and Bremer 2008).
data and did not include representatives from all genera.
We show that Coussarea–Faramea constitutes the sister
clade to remaining genera. Oreopolus and Cruckshanksia
have long been considered related based on morphology
(Taylor 1996), but few phylogenetic studies have included
Cruckshanksia. We confirm, with high support, the close
relationship between Oreopolus and Cruckshanksia. Heterophyllaea Hook.f. also belongs to this group (Andersson
and Rova, 1999), sister to the Oreopolus–Cruckshanksia
clade (Rydin et al. 2006). These three genera are all
restricted to the western parts of South America. The
Neotropical genera Coccocypselum and Declieuxia are
sisters and results from Rydin et al. (2006) highly support
the inclusion of Hindsia Benth. ex Lindl. in this clade, as
sister to Declieuxia. Piesschaert et al. (2000b) discussed
morphological as well as biogeographical support for the
Coccocypselum ? Declieuxia–Hindsia clade.
The tribe Danaideae is here sister to the Knoxieae–
Spermacoceae clade. The posterior probability for this
relationship is high, but the clade is collapsed in bootstrap
consensus trees. In Bremer and Manen (2000) Danaideae
was sister to the remaining Spermacoceae alliance (with
very low bootstrap support). More research is needed to
further assess the position of Danaideae.
Rubioideae
Conclusions
The sister relationship between Colletoecema dewevrei and
remaining Rubioideae is here confirmed with high support
(see also Robbrecht and Manen 2006; Rydin et al. 2008).
The next diverging clade comprises the East Asian
Ophiorrhizeae and the pantropical Urophylleae. This is
consistent with Andersson and Rova (1999), but the tribes
have otherwise often had an unresolved position at the base
of Rubioideae or they have formed a basal grade, being
subsequent sister groups to the rest of the subfamily
(Bremer and Manen 2000; Robbrecht and Manen 2006;
Razafimandimbison et al. 2008). The sister-group relationship between Ophiorrhizeae and Urophylleae is well
supported, but as often is the case for major groups in
Rubiaceae, obvious morphological support is difficult to
find.
Spiradiclis bifida, is here sister to Ophiorrhiza (Fig. 2),
but a rps16 sequence (Rydin et al. 2006) nested Spiradiclis
caespitosa Blume within Ophiorrhiza. The monophyly of
the two genera needs to be investigated further.
Coussareeae is a morphologically variable group,
restricted to the New World. Most species occur in lowland
rainforests, but the monotypic genus Oreopolus inhabits
the Andean regions. Several studies have contributed to our
understanding of relationships between the genera in
Coussareeae (Andersson and Rova 1999; Bremer and
Manen 2000), but they were based on the less amounts of
The systematic position of Acranthera, a long-debated
question, is resolved; Acranthera and Coptosapelta are
sisters. Acranthera is considered unique within Rubiaceae
in reproductive characters and obvious morphological
synapomorphies for the Acranthera–Coptosapelta clade
are currently not known, but the well-supported result in all
our analyses leaves little doubt about their close relationship. We performed a preliminary study of the pollen
grains of Acranthera in an attempt to find synapomorphies
with the unique pollen of Coptosapelta, but most characters
of Acranthera grains (for example size, the colporate grains
with three apertures positioned at angles and the reticulate
sexine) are common in Rubiaceae and probably plesiomorphic. There are some potential (derived) similarities
though; future studies may reveal new insights on morphological features of the clade.
Luculia is sister to Acranthera–Coptosapelta but the
clade is only well-supported in Bayesian analyses including nrITS. Nuclear ITS has traditionally been utilised
mainly for studying higher-level relationships, e.g. within a
genus, but it cannot be a priori assumed that homology
assessments are impossible for certain loci at certain taxonomic levels. Here, nrITS provided structured information
on deep divergences, as well as on higher-level relationships in Rubiaceae, and appear particularly useful in
Cinchonoideae and Ixoroideae.
before: Retiniphylleae sister to the (Vanguerieae–Ixoreae) ? (Alberteae–remaining Ixoroideae) clade (Fig. 1).
It should be noted, however, that no representatives of
Posoquerieae and Henriquezieae are included in the present
study. Further, Sipaneeae and Condamineeae are not sisters
but form a grade to remaining Ixoroideae in our ndhF
analyses and this is consistent with results found in
Kainulainen et al. (in press).
Cinchonoideae
123
120
Basal relationships within the three subfamilies Rubioideae, Cinchonoideae and Ixoroideae are indicated in
the present study, but deep divergences in the family were
not resolved. Single gene regions produced contradicting
(poorly supported) results and combined analyses resulted
in a basal polytomy consisting of (1) Luculia–Acranthera–
Coptosapelta, (2) an Ixoroideae-Cinchonoideae clade, (3)
Rubioideae. Like for example major relationships amongst
seed plants (Burleigh and Mathews 2007a, 2007b); mosses
and worts (Qiu et al. 2006); the position of Equisetum
(Schuettpelz et al. 2006) and relationships within the
angiosperm clades Ericales (Schoenenberger et al. 2005),
Lamiales (Wortley et al. 2005) and Malpighiales (APGII
2003), early radiation patterns within Rubiaceae have not
been unambiguously resolved despite that large amounts of
data have been analysed. In cases when molecular markers
produce conflicting results, other kinds of data, for example
structural rearrangements in the genomes, developmental
biology and comparative morphology, may be useful when
discriminating between alternative hypotheses.
Acknowledgments We thank the curators of the herbaria A.A.U.,
B.R., G.B., K., S., P. and U.P.S. for loan of herbarium material,
biomedical technicians Anbar Khodabandeh (Bergius Foundation,
Royal Academy of Sciences) and Keyvan Mirbakhsh (Stockholm
University, Sweden) for assistance, Jürg Schönenberger (Stockholm
University) for suggestions for improvement of SEM investigations,
Charlotte Taylor (Missouri Botanical Garden) for sharing unpublished
information on Dunnia sinensis, Peter Endress (University of Zürich),
Jan-Thomas Johansson, Per-Ola Karis (Stockholm University), Elmar
Robbrecht (National Botanic Garden, Belgium) and an anonymous
reviewer for valuable suggestions and comments on the text. The
study was supported by grants from the Swedish Research Council to
C.R. and B.B., and from the Knut and Alice Wallenberg Foundation
to B.B.
Appendix: FAMILY—RUBIACEAE JUSS.
Tribe Luculieae Rydin and B. Bremer, tribus nov.
Type: Luculia Sweet
Diagnosis: Arbuscula. Calyx 5-merous, corolla 5-mera,
tubo longo vix supra dilatato. Flores heterostyli. Antherae
intra tubum subsessiles semiexsertae. Stigmata 2, ovarium
2-loculare, loculis polyspermis. Fructus baccatus, semina
minuta.
Description: Small trees or shrubs, opposite phyllotaxis.
Stipules deciduous, lanceolate to linear. Flowers large,
showy, pentamerous, heterostylous. Stamens inserted in
narrow corolla tube, filaments short. Ovary bilocular, fruit
baccate, seeds small, numerous.
Genus included: Luculia Sweet
Useful publications: Murray (1990); Bremer et al.
(1999).
Tribe Coptosapelteae Bremek. ex S. Darwin, Taxon
25: p. 600, (Darwin 1976), emend. Rydin and B.Bremer
123
C. Rydin et al.
Type: Coptosapelta Kort.
Description: Sparsely branched subshrubs or vines.
Flowers usually pentamerous (rarely 4 or 6 parted). Ovary
bilocular, fruit a capsule, seeds numerous. Chromosome
basic number 10–11, Acranthera x10 (Kiehn 1995), Coptosapelta x11 (Verdcourt 1958; Puangsomlee and Puff
2001).
Note: The new circumscription is based on molecular
evidence presented in this paper. Morphological synapomorphies are not known at this point.
Genera included: Coptosapelta Kort., Acranthera Arn.
ex Meisn.
Useful publications: Alejandro et al. (2005); Verellen
et al. (2004); Puangsomlee and Puff (2001); Bremer et al.
(1999); Kiehn (1995); Puff et al. (1995); Bremekamp
(1947); Valeton (1923); Rydin et al. (this study).
SUBFAMILY—RUBIOIDEAE VERDC.
Bull. Jard. Bot. État Brux. 28: 280 (1958)
Tribe Colletoecemateae Rydin and B. Bremer, tribus
nov.
Type: Colletoecema E.M.A. Petit
Diagnosis: Arbores vel fructices, stipulis integris. Inflorescentiae axillares floribus multis conglomeratis. Flores
heterostyli, 5-meri. Calyx cupuliformis, corolla tubiformis,
stamina filamentis longis sub sinibus corollae adfixis.
Ovarium 2-loculare, ovulo 1. Fructus drupaceus, pyrena 2loculare, semina albumine satis molli et oleoso, embryo
teres.
Description: Small trees or shrubs. Inflorescences axillary, flowers pentamerous, heterostylous. Stamens inserted
in corolla tube. Ovary bilocular, one ovule per locule.
Embryo long and narrow. Fruit a drupe, pyrenes bilocular.
Genus included: Colletoecema E.M.A. Petit
Useful publications: Petit (1963); Piesschaert et al.
(2000a); Robbrecht and Manen (2006); Rydin et al. (2008).
Schizocoleeae Rydin and B. Bremer, tribus nov.
Type: Schizocolea Bremek.
Diagnosis: Arbuscula. Stipulae in vaginam longam et
angustam in fimbrias plerumque 8 fissam connatae. Flores
in axillis foliorum dispositi. Calyx 5-merous, lobis e basi
triangulari-setiformibus, hirsutis. Corolla hypocrateriformis, tubo calycem longitudine multo excedente. Stamina
parte dilatata tubi inserta. Ovarium biloculare, loculis septo
tenui separatis. Fructus baccatus, monospermus.
Description: Small trees, stipules bordered with fine hairs.
Flowers pentamerous, calyx triangular at base. Corolla
extends beyond calyx, stamens inserted in corolla tube. Ovary
bilocular with thin dissepiments separating the locules. Fruit a
berry, one-seeded, surmounted by persistent calyx.
Genus included: Schizocolea Bremek.
Useful publications: Bremekamp (1950); Razafimandimbison et al. (2008); Rydin et al. (2008).
Deep divergences in the coffee family and the systematic position of Acranthera
Dunnieae Rydin and B. Bremer, tribus nov.
Type: Dunnia Tutcher
Diagnosis: Frutex. Inflorescentiae terminales, cymosae,
floribus multis conglomeratis, bracteis magnis albis circumdatae. Flores 5-meri, calycis lobi minuti, persistentes.
Corolla tubiformis, tubo calycem longitudine multo excedente. Fructus capsularis, 2-valvis, valvis 2-partitis.
Semina numerosa.
Description: Woody shrubs, stipules pubescent. Inflorescences terminal cymes, surrounded by enlarged,
petaloid bracts. Flowers pentamerous, corolla tube extends
out of calyx. Stamens inserted in corolla lobe. Pistil
distylous. Fruit a capsule, seeds numerous. Diagnosis and
description are based on the original publication of Dunnia
(Tutcher 1905) and on observations made by C. Taylor
(Missouri Botanical Garden, pers. com.).
Genus included: Dunnia Tutcher
Useful publications: Tutcher (1905); Ge et al. (2002);
Chiang et al. (2002); Rydin et al. (2008).
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