See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/232273432

Molecular Phylogeny of Aristolochia sensu lato (Aristolochiaceae) based on

Sequences of rbcL, matK, and phyA Genes, with Special Reference to
Differentiation of Chromosome Numbers

Article  in  Systematic Botany · July 2006

DOI: 10.1600/036364406778388656

CITATIONS READS

81 866

6 authors, including:

Tetsuo Toma Stefan Wanke

Okayama University of Science Technische Universität Dresden
58 PUBLICATIONS   440 CITATIONS    195 PUBLICATIONS   2,829 CITATIONS   

SEE PROFILE SEE PROFILE

Jin Murata
The University of Tokyo
199 PUBLICATIONS   3,155 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Field Guide to Aquatic Plants of Myanmar View project

Ginkgo biloba View project

All content following this page was uploaded by Stefan Wanke on 28 May 2014.

The user has requested enhancement of the downloaded file.

Systematic Botany (2006), 31(3): pp. 481–492
! Copyright 2006 by the American Society of Plant Taxonomists

Molecular Phylogeny of Aristolochia sensu lato (Aristolochiaceae) based on

Sequences of rbcL, matK, and phyA Genes, with Special Reference to
Differentiation of Chromosome Numbers

TETSUO OHI-TOMA,1,5 TAKASHI SUGAWARA,2 HIROKO MURATA,3 STEFAN WANKE,4

CHRISTOPH NEINHUIS,4 and JIN MURATA1
Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo 112-0001, Japan;
1

2
Makino Herbarium, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan;
3
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan;
4
Institut für Botanik, Technische Universität Dresden, 01062 Dresden, Germany
5
Author for correspondence (ooi@ns.bg.s.u-tokyo.ac.jp)

Communicating Editor: Paul S. Manos

ABSTRACT. The genus Aristolochia sensu lato contains over 400 species from warm temperate to tropical regions world-
wide. Taxonomic treatments of Aristolochia have been ambiguous and controversial. In a recent cladistic analysis based on
morphological characters, it was proposed that the genus should be divided into four genera in two subtribes. To reconsider
the systematics of Aristolochia sensu lato, we reconstructed its phylogeny based on nucleotide sequences of the chloroplast
rbcL gene and the nuclear-encoded phytochrome A (phyA) gene for 19 representative species and the chloroplast matK gene
of over 80 species. All phylogenetic trees produced with the three genes indicate that Aristolochia sensu lato is a monophyletic
group, consisting of two lineages that correspond to the subtribes Aristolochiinae and Isotrematinae. The matK phylogeny
shows that each of the lineages includes two sublineages. The Aristolochiinae clade is composed of the Aristolochia sensu
stricto and Pararistolochia clades, and the Isotrematinae clade of the Isotrema and Endodeca clades. Chromosome numbers,
including newly reported counts for 30 species, are predominantly congruent with the phylogeny: the Aristolochiinae clade
shows chromosome numbers of 2n ! 6, 12, 14, or 16, while the Isotrematinae clade is characterized by 2n ! 32. In the
Isotrematinae clade, the paralogous relationships of the phyA gene suggest that polyploidization might have occurred.
KEYWORDS: Aristolochia, chromosome numbers, matK, phyA, phylogeny, rbcL.

The genus Aristolochia L. sensu lato (s.l.) is the larg-
est and most diverse genus of the family Aristolochi-
aceae, containing over 400 species from temperate to
tropical regions worldwide. Most species of Aristolochia
s.l. are lianas, some are perennial herbs, and a few are
shrubs. All have zygomorphic tubular flowers with a
gynostemium. In distinguishing taxa, the morphology
of the perianth, gynostemium, and fruit are commonly
regarded as useful characters. However, the circum-
scription of the genus has been a significant taxonomic
problem (reviewed by González and Stevenson 2002).
In some treatments, a single genus Aristolochia s.l., con-
sisting of three subgenera, Aristolochia (! Orthoaristo-
lochia), Pararistolochia (Hutchinson & Dalziel) O. C.
Schmidt, and Siphisia (Raf.) Duch., has been recognized
(Schmidt 1935; Ma 1989; González 1999; González and
Stevenson 2000). Hutchinson and Dalziel (1954), Poncy
(1978), and Parsons (1996) recognized two genera,
Aristolochia and Pararistolochia Hutchinson & Dalziel,
and Klotzsch (1859) recognized five genera. In con-
trast, Huber (1993) recognized eight genera in two
subtribes, Aristolochia sensu stricto (s.s.), Einomeia Raf.,
Euglypha E. Chodat & Hassl., Holostylis Duch., Howardia
Klotzsch, and Pararistolochia in the subtribe Aristolo-
chiinae H. Huber, and Endodeca Raf. and Isotrema Raf.
in the subtribe Isotrematinae H. Huber.
Recently, González and Stevenson (2002) examined
the relationships within Aristolochia s.l. based on a cla-
481
distic analysis of a number of morphological charac-
ters. Their results showed that Einomeia, and the mono-
typic Euglypha and Holostylis, which were accepted as
distinct genera by Huber (1993), were nested within
Aristolochia s.s. They also indicated that the genus sensu
lato consists of two major lineages that were congruent
with the subtribes Aristolochiinae and Isotrematinae
of Huber (1993). They proposed that Aristolochia s.l.
should be divided into four genera in two subtribes:
Aristolochia s.s. and Pararistolochia in Aristolochiinae,
and Endodeca and Isotrema in Isotrematinae. Kelly and
González (2003) substantiated the data, and also dem-
onstrated closer relationships between Aristolochia s.s.
and Pararistolochia, and between Endodeca and Isotrema
based on a more detailed cladistic analysis of Aristo-
lochiaceae. Comparing this system with that in which
three subgenera of Aristolochia s.l. are recognized, Aris-
tolochia s.s. and Pararistolochia of the subtribe Aristo-
lochiinae correspond to the subgenera Aristolochia and
Pararistolochia respectively, and the subtribe Isotrema-
tinae is congruent with the subgenus Siphisia. Thus,
the taxonomic frameworks are largely identical in sev-
eral systems, but the recognitions of their taxonomic
ranks are different. Until now, the phylogeny and clas-
sification of Aristolochia s.l. has been based mainly on
morphological characters and has relied only slightly
on molecular data and chromosome numbers.
Previous molecular studies of Aristolochia s.l. using
482 SYSTEMATIC BOTANY
the chloroplast matK gene for ca. 60 species revealed
that the genus is monophyletic, and includes two dis-
tinct lineages and several sublineages (Murata et al.
2001). The results were nearly congruent with the clas-
sification of González (1999), showing the reciprocal
monophyly of the two subgenera Aristolochia and Si-
phisia, and the monophyly of most sections, subsec-
tions, and series in the former subgenus. Unfortunate-
ly, this molecular phylogeny lacked species of the sub-
genus Pararistolochia, which is often recognized as a
distinct genus, and several of Huber’s smaller genera,
such as Einomeia, Endodeca, Euglypha, and Holostylis.
More recently, Neinhuis et al. (2005) presented a
molecular phylogeny that included Pararistolochia, Ei-
nomeia, Endodeca, and Holostylis, based on cpDNA se-
quences of the trnL intron and the intergenic spacer
region between the trnL 3" exon and the trnF gene.
Their phylogeny was congruent with the system of
González and Stevenson (2002), but they sampled only
29 of 400 species of Aristolochia s.l. and included only
a few Asian or South American species. Because they
focused on the higher relationships in Aristolochiaceae,
their study did not clarify the phylogenetic relation-
ships and classification within Aristolochia s.l.
Chromosome number is a valuable character to
characterize clades in Aristolochia s.l. Sugawara et al.
(2001) showed that the distribution of chromosome
numbers is also congruent with the system of Gon-
zález (1999). Subgenus Aristolochia presents a diversity
of chromosome numbers, with 2n ! 12, 14, or 16; 2n
! 12 and 2n ! 16 are predominantly specific to sub-
section Podanthemum (Klotz.) Schmidt and to series
Thyrsicae F. González, respectively. Chromosome num-
bers 2n ! 28 and 2n ! 32 have been reported for sub-
genus Siphisia (Gregory 1956; Ma 1989; Goldblatt and
Johnson 1991; Yu et al. 1995), although the former
number is considered to be a miscount (Sugawara et
al. 2001). It was thought that subgenus Siphisia has an
ancient polyploid origin. The chromosome number of
Pararistolochia has been unknown until now (Huber
1993; González and Stevenson 2000).
In this study, we examined phylogenetic relation-
ships within Aristolochia s.l., especially the relation-
ships of the four major groups proposed both by Gon-
zález and Stevenson (2002) based on morphological
data and by Neinhuis et al. (2005) based on molecular
data. Initially, to clarify the chromosome numbers and
compare with the phylogenetic relationships in Aris-
tolochia s.l., chromosomes of 31 samples of 30 species
were newly examined. We then constructed a detailed
phylogeny of Aristolochia s.l. based on the nucleotide
sequences of the chloroplast rbcL and matK genes and
the nuclear-encoded phytochrome A (phyA) gene of the
phytochrome gene family. We examined the phyloge-
netic relationships among the major genera and sub-
genera using rbcL and phyA sequences from 19 repre-
[Volume 31
sentative species of Aristolochia s.l., selected on basis of
their generic or infrageneric placements as proposed
by Huber (1993), González (1999), and González and
Stevenson (2002) and their chromosome numbers. We
then performed a phylogenetic analysis based on the
nucleotide sequences of the matK gene of 98 accessions
for 85 species, to examine the phylogenetic relation-
ships among sections, and species in more detail.
MATERIALS AND METHODS
Taxon Sampling. The generic status of the samples was pre-
liminarily regarded as Aristolochia s.l. Most of the samples used in
previous studies (Murata et al. 2001; Sugawara et al. 2001; Nein-
huis et al. 2005), and 34 new samples were used (Appendix 1). Of
these, six samples (Aristolochia spp. JM01 to JM06) are still un-
identified, and four samples are treated as similar to a named
species (‘‘aff.’’), although it will be necessary to examine whether
they are new species. Unfortunately, we could not obtain material
of Aristolochia rojasiana (Chodat & Hassl.) F. González, recognized
as the monotypic genus Euglypha by Huber (1993).
The Aristolochiaceae comprise four major genera, Aristolochia
s.l., Thottea Rottb. s.l., Asarum L. s.l., and Saruma Oliver (González
1999; González and Stevenson 2000). Representative species of the
latter three genera and Lactoris fernandeziana Phil. (Lactoridaceae)
were used as outgroups in all analyses, in accordance with the
recent phylogenies of the angiosperms (Qiu et al. 1993, 1999; Sa-
volainen et al. 2000; Soltis et al. 2000; Nickrent et al. 2002; APG
2003; Borsch et al. 2003; Zanis et al. 2003; Jaramillo et al. 2004;
Neinhuis et al. 2005). The nucleotide sequences for the outgroup
species were determined in this study or obtained from GenBank.
However, the rbcL sequence data of Thottea tomentosa (BI.) Ding
Hou obtained from GenBank (AF197598) was more similar to our
sequence of Aristolochia arborea Duch. than to that of T. tomentosa.
Because its identity was doubtful, we did not use this sequence in
our analysis.
Chromosome Observation. The chromosomes of 31 samples
from 30 species, including three species that are often treated as
Pararistolochia (A. praevenosa, A. promissa and A. triactina), were
newly examined. Species that have been reported to be 2n ! 28
(A. californica, A. serpentaria, A. tomentosa) were thoroughly reex-
amined. Plant materials were obtained from transplants of natural
populations and from cultivation at the Botanical Gardens of the
University of Tokyo, and the Medical Garden of Setsunan Univer-
sity. We observed and counted the somatic chromosomes in root
tip cells by microscopy following the procedures of Sugawara et
al. (2001).
DNA Extraction, Amplification and Sequencing. Total genomic
DNA was extracted from silica-gel dried leaf tissue using the
method of Doyle and Doyle (1987), with slight modifications. Se-
quences determined in the present analysis were registered with
the DNA Data Bank of Japan (DDBJ), which is linked to GenBank,
and their accession numbers are given with the sample informa-
tion in Appendix 1.
The nucleotide sequences of the chloroplast rbcL and matK genes
were determined by standard polymerase chain reaction (PCR)
and direct sequencing methods. For the 19 representative species,
the rbcL gene was amplified using four primers: 1F and 636F (Fay
et al. 1997), and 729R (5"- CTT CGC ATG TAC CTG CAG TAG
C#3") and R23 (5"- TTT TAG TAA AAG ATT GGG CCG#3") (T.
Kajita, pers. comm.). The PCR reaction mixture consisted of 1.0
unit of ExTaq DNA polymerase (TaKaRa Bio, Ohtsu, Shiga, Japan),
3 $L of 10% ExTaq buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl,
1.5 mM MgCl2), 2.4 $L of 0.2 mM dNTP solution, 1.0 $L of each
10 pM primer, and 10–30 ng of genomic DNA, in a total volume
of 30 $L. PCR cycling conditions were 96%C for 45 sec, then 30
cycles of 96%C for 45 sec, 52%C for 45 sec, 72%C for 1 min, and
finally 72%C for 10 min. For the new 34 samples, the matK gene
was amplified following the method of Murata et al. (2001), with
minor modifications. The PCR products were purified using the
2006] OHI-TOMA ET AL.: ARISTOLOCHIA MOLECULAR PHYLOGENY
GeneClean III DNA Purification Kit (BIO 101, Carlsbad, CA, USA),
following the instructions provided by the manufacturer. Purified
template DNA fragments were amplified using the ABI PRISM Big
Dye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA)
with the same primers as those used for PCR. DNA sequencing
was performed using an ABI PRISM 377 DNA sequencer (Applied
Biosystems). The program AutoAssembler (Applied Biosystems)
was used to assemble complementary sequences.
Fragments of phyA were initially amplified by PCR using the
locus-specific primer set of Mathews and Donoghue (1999), PHYA-
upstream and PHYA-downstream, for the 19 representative spe-
cies. The PCR cycling conditions were 96%C for 45 sec, then 28
cycles of 96%C for 45 sec, 55%C for 45 sec, 72%C for 1 min, and
finally 72%C for 10 min. The fragments obtained were purified and
directly sequenced, and then new primer sets were designed for
Aristolochiaceae: PHYA-AristoF (5"- DCA TTA CCC RGC CAC
AGA#3") and PHYA-AristoR (5"- CTM ACD GCW GCC ATA CCA
CA#3"), which amplified fragments approximately 700 bp long,
using the primer set under the same conditions as the previous
PCR. Direct sequencing of the PCR products from eight species
showed overlapping double peaks or superimposed waves at the
same sites for both complementary strands on the program
AutoAssembler. These products were cloned into the pCR 4-TOPO
vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen
Corp., Carlsbad, CA, USA). Eight to 10 clones per sample were
picked from clones grown on Luria-Bertani agar plates containing
ampicillin. The plasmids, including the target fragment of the
phyA gene, were amplified by the rolling cycle amplification (RCA)
method using the TempliPhi DNA Amplification Kit (Amersham
Biosciences Corp., Piscataway, NJ, USA), following the instructions
provided by the manufacturer. Using the RCA products, cycle se-
quencing was performed with the same primers as those used for
PCR amplification. Mutations at the attachment site of PHYA-
AristoF were found only in the sample of A. kaemferi Willd, so ca.
950 bp product was amplified with a combination of PHYA-AristoR
and PHYA-downstream, and was cloned and sequenced.
Phylogenetic Analysis. To examine the monophyly of the ge-
nus Aristolochia s.l., and to clarify the phylogenetic relationships
among the major groups of the genus, analyses based on rbcL and
phyA sequences were conducted for representative species. More
detailed infrageneric relationships throughout Aristolochia s.l. were
then inferred based on nucleotide sequences of the matK gene in
all samples.
The multiple sequences of each gene were initially aligned with
the CLUSTAL X program (Thompson 1997) and then adjusted
manually. The data matrices are available from TreeBASE (study
accession number S1531). Phylogenetic analyses were conducted
using the maximum parsimony (MP) and maximum likelihood
(ML) methods of PAUP* 4.0b10 (Swofford 2002). In the MP anal-
yses of the rbcL and phyA genes, only substitutions were used
because no length polymorphisms occurred in the final matrix. In
the MP analysis of the matK gene, nucleotide substitutions and
length polymorphisms, coded as a fifth state, were equally weight-
ed. To search for the shortest trees, MP analysis was performed
using a heuristic search with 100 replicates of random sequence
addition, tree-bisection-reconnection branch swapping, and the
Multrees option. The limit of MaxTrees option was set at no limits
in the analyses of rbcL and phyA genes, but at 50,000 trees in each
random addition sequence replicate in the matK analysis. The strict
consensus tree of the most parsimonious trees was generated, and
then character changes were reconstructed on the tree with ACCT-
RAN character optimization using PAUP. Bootstrap analyses were
performed using 1,000 replicates and the same tree search proce-
dure as described above, except with the simple addition sequenc-
es. For the matK data set, the upper limit of MaxTrees was set to
1,000 trees in the bootstrap analysis.
All ML analyses were based on substitutions only using the best
model identified and parameter values estimated by the hierar-
chical likelihood ratio test in the program Modeltest 3.06 (Posada
and Crandall 1998). ML heuristic searches were conducted using
10 replicates with random sequence addition. Due to the enormous
amount of time required for bootstrap analyses using the ML
483
method, we used neighbor-joining bootstrap analysis with ML
distances to approximate bootstrap support for the ML trees, fol-
lowing Fan and Xiang (2003). The same substitution model and
parameters used in the ML tree search were used in the ML dis-
tance estimations. Ten thousand bootstrap replicates were per-
formed.
RESULTS
Chromosome Numbers. Counts of somatic chro-
mosomes are given in Table 1. Five different chromo-
some numbers, 2n ! 6, 12, 14, 16, and 32, were ob-
served (Fig. 1; Table 1). All but 2n ! 6 have been re-
ported in the genus in previous studies (Nardi 1984;
Sugawara et al. 2001), but the count of 2n ! 6 for A.
burelae as reported here for the first time is the lowest
chromosome number in Aristolochia s.l. The chromo-
some numbers (2n ! 12) of species often placed in the
genus Pararistolochia (Aristolochia praevenosa, A. promis-
sa, and A. triactina) also are reported here for the first
time. Their chromosomes and those of A. burelae are
relatively larger than those of other species of Aristo-
lochia s.l. Moreover, the chromosomes of A. promissa
and A. triactina from Africa are larger than the others
(Fig. 1).
rbcL Phylogeny. The rbcL sequences generated for
Aristolochia s.l. varied from 1,417 to 1,425 bp in length.
Two samples of A. micrantha and A. pentandra have the
same insertion of eight bases adjacent to the 3" end.
The insertion is located in the flanking non-coding re-
gion and a stop codon occurs upstream from the in-
sertion. About 250 bp of the nucleotide sequence from
the 3" end of the fragment, including this insertion,
was omitted from the phylogenetic analysis, because
the lengths of some sequences obtained from GenBank
were shorter than those of Aristolochia (e.g., Lactoris fer-
nandeziana, L08763). After the multiple sequences were
aligned, a total aligned length of 1,179 bp formed the
final matrix. The MP analysis resulted in 35 most par-
simonious trees of 278 steps, with CI ! 0.719, RI !
0.860, and RC ! 0.619. In the ML analysis, the Hase-
gawa-Kishino-Yano model (Hasegawa et al. 1985) with
a gamma distribution and proportion of invariant sites
(the HKY&G&I model) was selected as the best fitting
model for the rbcL sequence data, and the best likeli-
hood score for the ML tree obtained was #ln !
3396.54793. Because the topologies of the strict consen-
sus MP tree and the ML tree were largely identical,
the strict consensus (MP) tree is shown in Fig. 2A.
Aristolochia s.l. is monophyletic and sister to Thottea.
It consists of two strongly supported major clades that
correspond to the subtribes Aristolochiinae and Isotre-
matinae of Huber (1993) and González and Stevenson
(2002). In the clade Aristolochiinae, several subclades
are formed but their relationships are unresolved.
Three species, A. praevenosa, A. promissa, and A. triac-
tina, which have been often treated as Pararistolochia,
form a monophyletic group with relatively strong sup-
484 SYSTEMATIC BOTANY [Volume 31

TABLE 1. Somatic chromosome numbers of samples examined in the present study. The generic status of all samples is regarded as
Aristolochia s.l., and species are categorized into our proposed genera and subgenera. Voucher specimens of A. promissa and A. triactina
are kept in BONN, and others in TI.

Chromosome
Species number (2n) Voucher Source

Aristolochia subgen. Aristolochia

A. maxima 16 Murata et al. SETS37 cultivation
A. anguicida 14 Murata et al. JM03–140 cultivation
A. cauliflora 14 Murata et al. SETS85 Malaysia
A. esperanzae 14 Murata et al. SETS88 cultivation
A. littoralis 14 Murata et al. JM04–45 cultivation
A. peruviana 14 Murata et al. SETS70 cultivation
A. paulistana 14 Murata et al. SETS91 Brazil
A. pilosa 14 Murata et al. SETS6 Honduras
A. pilosa 14 Murata et al. SETS90 Costa Rica
A. tentaculata 14 Murata et al. SETS87 Mexico
A. sp. (JM02) 14 Murata et al. SETS49 India, Assam
A. sp. (JM04) 14 Murata et al. SETS90 Myanmar
A. sp. (JM06) 14 Murata et al. JM04–46 Brazil
A. gaudichaudii 12 Murata et al. SETS8 Indonesia
A. hookeriana 12 Murata et al. JM00–2 Myanmar
A. indica 12 Ohi-Toma & Sugawara 040514 Myanmar
A. kerrii 12 Murata et al. JM02–260 Myanmar
A. tagala 12 Ohi-Toma & Sugawara 040521 Myanmar
A. taliscana 12 Murata et al. SETS79 Mexico
A. burelae 6 Murata et al. JM00–1 Bolivia
Aristolochia subgen. Pararistolochia
A. praevenosa 12 Koishikawa BG, Ohi-Toma s.n. Australia
A. promissa 12 BG Bonn 13014, Neinhuis 118 Africa
A. triactina 12 BG Bonn 12767, Neinhuis 119 Africa
Isotrema subgen. Isotrema
A. arborea 32 Murata et al. JM99–1 cultivation
A. californica 32 Sugawara et al. SETS76 USA
A. mollissima 32 Murata et al. SETS30 China
A. neolongifolia 32 Murata et al. SETS96 China
A. saccata 32 Murata et al. SETS86 Myanmar
A. tomentosa 32 Murata et al. SETS77 USA
A. wardiana 32 Murata et al. SETS98 Myanmar
Isotrema subgen. Endodeca
A. serpentaria 32 Murata et al. SETS84 USA

port. Both A. holostylis and A. pentandra, which have
been previously treated as Holostylis and Einomeia, re-
spectively, by Huber (1993), are nested within the Aris-
tolochia s.s. clade. Aristolochia holostylis forms a clade
with A. gigantea and A. eriantha. Aristolochia pentandra
is closely related to A. micrantha and this clade is sister
to A. burelae. The Isotrematinae clade contains two sub-
clades with strong supports: one consisting of the two
species, A. serpentaria and A. convolvulacea, which be-
long to Endodeca, and the other including the species
placed in Isotrema (sensu Huber 1993; González and
Stevenson 2002).
phyA Phylogeny. The phyA sequences generated
for Aristolochia s.l. varied from 700 to 715 bp in length.
The sequences determined could be translated into
amino acids without stop codons, except for two se-
quences, one each from A. micrantha and A. pentandra.
Compared to the complete sequence of the phyA gene
of Arabidopsis thaliana (GenBank accession L21154), the
sequences we obtained in our analysis were homolo-
gous to one part of the exon 2 region of the phyA gene.
After multiple sequence alignments including those of
all outgroup taxa, the total aligned length of the phyA
data set was 721 bp. Parts of the alignment that were
ambiguous were excluded from the analysis, and a to-
tal of 714 bp formed the final matrix. The MP analysis
resulted in one most parsimonious tree of 634 steps
with CI ! 0.670, RI ! 0.772, and RC ! 0.517 (Fig. 2B).
In the ML analysis, the Hasegawa-Kishino-Yano model
(Hasegawa et al. 1985) with a gamma distribution (the
HKY&G model) was selected as the best fitting model
for the phyA sequence data, and the best likelihood
score for the obtained ML tree was #ln ! 4226.05120.
The topologies of the MP and ML trees differ in the
placement of A. praevenosa. In the MP tree, the species
is sister to the remaining taxa, and in the ML tree, A.
2006] OHI-TOMA ET AL.: ARISTOLOCHIA MOLECULAR PHYLOGENY 485

FIG. 1. Somatic chromosome numbers of representatives of Aristolochia sensu lato. A, A. burelae (2n ! 6); B, A. tagala (2n !
12); C, A. praevenosa (2n ! 12); D, A. triactina (2n ! 12); E, A. promissa (2 n ! 12); F, A. argentina (2n ! 14); G, A. ovalifolia (2 n
! 16); H, A. wardiana (2 n ! 32). Details of the samples are given in Table 1, excepting F and G from Sugawara et al. (2002).
Scale bar is a length 10 $m.

promissa and A. triactina are sister to the remaining
taxa. However, the branch support values for both
placements are low.
The MP tree indicates that Aristolochia s.l. is mono-
phyletic and consists of two major lineages with strong
support, consistent with the results of the rbcL tree,
and members of each lineage are congruent with those
of each lineage in the rbcL tree (Fig. 2). One lineage
comprises samples with the heterogeneous sequences
of A. convolvulacea, A. serpentaria, A. arborea, A. kaemp-
feri, and A. tomentosa in the subtribe Isotrematinae.
Their sequences show a paralogous relationship and
the topologies of the paralogues, designated paralogue
1–1 and 1–2, are identical. Each comprises two sub-
clades: one containing A. serpentaria and A. convolvu-
lacea corresponding to Endodeca, and the other contain-
ing the species of Isotrema, sensu Huber (1993) and
González and Stevenson (2002).
The other lineage is congruent with subtribe Aris-
tolochiinae. Several subclades, A. albida & A. zollinger-
iana & A. clematitis; A. gigantea & A. eriantha & A. ho-
lostylis; and A. burelae & A. micrantha & A. pentandra,
matched those detected in the rbcL tree. Three species
of Pararistolochia, however, do not form a monophyletic
group, and are sister to the remaining taxa. The Aris-
tolochiinae clade also includes four samples with two
heterogeneous sequences. Of these, A. eriantha and A.
holostylis, which occur in the same subclade, have the
two most similar sequences. Aristolochia micrantha and
A. pentandra have two paralogous sequences, para-
logues 2–1 and 2–2, in contrast to the sister sequence
of A. burelae. Furthermore, the sequences of paralogue
486 SYSTEMATIC BOTANY [Volume 31

FIG. 2. (A) Strict consensus tree of 35 most parsimonious trees, based on sequences of the chloroplast rbcL gene (length !
278 steps, CI ! 0.719, RI ! 0.860, RC ! 0.619). (B) Single most parsimonious tree based on sequences of the nuclear-encoded
phyA gene (length ! 634 steps, CI ! 0.670, RI ! 0.772, RC ! 0.517). Numbers on branches are bootstrap values (over 50%)
estimated by MP analysis, and those in parentheses are bootstrap values (over 50%) estimated by NJ analysis based on ML
distances. Species names with accession numbers in parentheses are cited from GenBank. The subtribes and genera proposed
based on the morphological analysis of González and Stevenson (2002) are indicated at the nodes of the clades and between
the two trees, respectively. In the phyA phylogeny, the solid bars on the branch represent one-base deletions. Open circles at
the nodes represent putative polyploidization events. Gray bars on the left side of the tree represent paralogues of the phyA
gene.

2–2 have a two base deletion and a one base deletion
in each species. In these sequences, a stop codon is
introduced after the deletion by a frameshift, and these
paralogues may be pseudogenes.
matK Phylogeny. To examine the phylogenetic re-
lationships among species of Aristolochia s.l. in detail,
we analyzed the matK gene sequences of all samples.
The matK sequences generated for Aristolochia s.l. var-
ied from 1,194 to 1,224 bp in length. A total aligned
length of 1,281 bp, including the outgroup sequences,
formed the final data set. Nine length polymorphisms
with unambiguous alignment were treated as fifth
state characters in the MP analysis. The strict consen-
sus of 50,000 MP trees (the upper limit of MaxTrees;
1,017 steps, CI ! 0.613, RI ! 0.892, RC ! 0.547) and
the ML tree with #ln ! 7732.32404, derived by the
transversion model with a gamma distribution and
proportion of invariant sites (the TVM&G&I model)
differ in branching patterns among some species. The
ML tree shows greater resolution, but support for these
regions is weak. Because the clades with strong sup-
port detected in both trees are identical, the MP strict
consensus tree is shown in Fig. 3.
The tree supports the results of the rbcL and phyA
analyses showing that Aristolochia s.l. is monophyletic
and comprises two major clades, the Isotrematinae and
Aristolochiinae clades. In the Isotrematinae clade, both
Endodeca and Isotrema of Huber (1993) are strongly
supported as monophyletic, and the number of steps
separating the Endodeca and Isotrema clades is relatively
great (Fig. 3). The Endodeca clade consists of only two
herbaceous species, A. serpentaria and A. convolvulacea
from North America. The Isotrema clade contains spe-
cies from North and Central America to East Asia. A
group of shrubby species, A. arborea, A. salvadorensis,
and A. tricaudata, is a sister group to the woody liana,
A. paracleta of Central America. Among the other
woody lianas, the North American species and the
East Asian species are not clustered by geographical
region: Aristolochia tomentosa and A. macrophylla of
North America are sisters to A. manshuriensis of East
2006] OHI-TOMA ET AL.: ARISTOLOCHIA MOLECULAR PHYLOGENY
Asia, and A. californica of North America is nested
within the Asian clade of A. nakaoi to A. shimadai.
In the Aristolochiinae clade, Aristolochia s.s. (includ-
ing Einomeia and Holostylis of Huber 1993) and Parar-
istolochia form monophyletic groups (Fig. 3). However,
branch lengths for these clades are relatively short.
Several subclades are detected in the Aristolochia s.s.
clade, but their relationships are unclear. Using the
classification system of González and Stevenson (2002),
the two sections Diplolobus and Gymnolobus are not re-
solved as a clade. However, several subclades are
largely congruent with the infrageneric taxa. The re-
ciprocal monophyly of subsections Aristolochia and Po-
danthemum (Klotz.) Duch. of section Diplolobus are
strongly supported. In section Gymnolobus, subsection
Pentandrae Duch. appears to be monophyletic, but sub-
section Hexandrae is not. In subsection Hexandrae, series
Thyrsicae F. González is monophyletic but series Hex-
andrae is not, because A. holostylis is nested within the
clade of series Hexandrae, A. burelae appears as sister
to subsection Pentandrae, and A. grandiflora forms a
clade with subsections Aristolochia and Podanthemum,
although support for these clades is not strong.
Chromosome Numbers versus Phylogeny. Chro-
mosome numbers, which had been reported in previ-
ous studies or were newly determined in the present
study, were compared with the matK phylogeny, al-
though some were unknown (Fig. 3). Previous studies
have attributed a chromosome number of 2n ! 28 to
some species of Isotrematinae (Gregory 1956; Ma 1989;
Goldblatt and Johnson 1991; Yu et al. 1995), but Su-
gawara et al (2001) questioned those reports because
they were unable to find 2n ! 28 among their samples.
We observed only 2n ! 32 in those species of Isotre-
matinae that we were able to examine, and consequent-
ly also doubt the chromosome counts of 2n ! 28 in
species of Isotrematinae. Therefore we did not include
in our analysis the chromosome number of 2n ! 28.
The Isotrematinae clade is characterized by the chro-
mosome number 2n ! 32. In the Aristolochiinae clade,
subclades are characterized by different numbers of
chromosomes. The chromosome numbers of species of
the Pararistolochia clade and the subsection Podanthe-
mum clade are 2n ! 12, those of the subsection Aris-
tolochia clade and the subsection Pentandrae clade are
2n ! 14, and those of series Thyrsicae clade are 2n !
16. The clade consists of most species of series Hex-
andrae (except A. burelae) is characterized by 2n ! 14.
Aristolochia burelae, which has the lowest chromosome
number (2n ! 6), is sister to the subsection Pentandrae
clade, which has 2n ! 14 chromosome.
DISCUSSION
Chromosome Numbers and Phylogeny. In the pre-
sent study, five different chromosome numbers (2n !
6, 12, 14, 16, and 32) were identified in Aristolochia s.l.
487
(Fig. 1; Table 1). Nardi (1984) reported that species of
the subtribe Aristolochiinae from the Mediterranean
region display various chromosome numbers, 2n ! 8,
10, 12, 14, 16, 24, 26, 28, and 36. In the species exam-
ined here, we did not observe most of those numbers
because we lacked most of the species from that re-
gion. Those chromosome numbers may be specific to
species from the Mediterranean region. Considering
the numbers reported by Nardi (1984), this is the first
report of the chromosome number 2n ! 6, identified
in A. burelae, which is the lowest number yet observed
in Aristolochia s.l.
The chromosome numbers of Aristolochia s.l. corre-
spond closely to the molecular phylogeny (Fig. 3). The
Isotrematinae clade is characterized by the highest
number of chromosomes, 2n ! 32, whereas the Aris-
tolochiinae clade includes species with several differ-
ent chromosome numbers, 2n ! 6, 12, 14, and 16. In
the latter clade, the subclades of Pararistolochia (2n !
12), subsection Aristolochia (2n ! 14), subsection Po-
danthemum (2n ! 12), subsection Pentandrae (2n ! 14),
and series Thyrsicae (2n ! 16) are characterized by spe-
cific chromosome numbers. However, subclades shar-
ing the same chromosome number are not always
monophyletic.
Of the three genes analyzed, the nuclear-encoded
phyA gene provides additional useful information re-
garding chromosomal evolution in Aristolochia s.l.. The
gene is reported as a single copy gene in A. grandiflora
(Mathews and Donoghue 2000), which has 2n ! 14
and is a member of the Aristolochiinae clade. Our
analysis found two copies and paralogous relation-
ships in some groups of Aristolochia s.l. (Fig. 2B). Spe-
cies with phyA paralogues belong to the Isotrematinae
clade, the members of which have more chromosomes
(2n ! 32) than do the species of the Aristolochiinae
clade (2n ! 6, 12, 14, or 16). It is inferred that genome
duplication by polyploidization in the common ances-
tor of the Isotrematinae clade resulted in the duplica-
tion of the phyA gene. Previous studies of the homo-
logues of the B-class genes APETALA3 (AP3) and PIS-
TILLATA (PI) also suggested a polyploidization event
in the subtribe Isotrematinae (Stellari et al. 2004; Jar-
amillo and Kramer 2004). One homologue of each gene
was isolated in A. eriantha of the subtribe Aristolochi-
inae, although its chromosome number was unknown.
Aristolochia manshuriensis of the subtribe Isotrematinae,
with 2n ! 32, has two paralogues of both genes.
In the Aristolochiinae clade, species of the Pentandrae
clade with 2n ! 14, equal to the chromosome number
of A. grandiflora, also have paralogues of the phyA gene
(Fig. 2B). The gene duplication observed in the Pentan-
drae clade, which is sister to A. burelae with 2n ! 6,
may be the result of polyploidization, with subsequent
changes in chromosome number.
Subfamily Aristolochioideae. Kelly and González
488 SYSTEMATIC BOTANY [Volume 31

FIG. 3. Strict consensus tree of 50,000 most parsimonious trees (the upper limit in the MaxTree option), based on sequences
of the chloroplast matK gene (length ! 1,017 steps, CI ! 0.613, RI ! 0.892, RC ! 0.547). Numbers on the branches are bootstrap
values (over 50%) estimated by MP analysis, and those in parentheses are bootstrap values (over 50%) estimated by NJ analysis
based on ML distances. Numbers in parentheses with species name are somatic chromosome numbers, and asterisks indicate
species with previously reported chromosome of 2n ! 28. Chromosome numbers (2n) in clades are summarized on the right
side of the tree. The geographic range of species (Dist.) is also indicated on the right side of the tree. Abbreviations of areas:
N.Am ! North America, C.Am ! Central America, S.Am ! South America, Eu ! Europe, As ! Asia, E.As ! East Asia, Aust
! Australia, Af ! Africa, Mad ! Madagascar, Bo-Br-P ! Bolivia, Brazil and Peru. Bold branches on the tree indicate the clades,
which we propose as genera or subgenera. The subtribes and genera proposed based on the morphological analysis of González
and Stevenson (2002) are also indicated on the nodes of the clades. The infrageneric ranks of González (1999) and the small
genera of Huber (1993) are shown on the right of the Aristolochia s.s. clade.

(2003) summarized the traditional framework of the
subfamilies of Aristolochiaceae, emphasizing the tax-
onomic position of Thottea s.l., a genus belonging to
the subfamily Asaroideae or Aristolochioideae. They
agreed with the system of Huber (1993), in which Thot-
tea s.l. is sister to Aristolochia s.l. in subfamily Aristo-
lochioideae, as determined by a cladistic analysis based
on morphological characters. Neinhuis et al. (2005)
also supported Huber’s system based on data from the
chloroplast trnL—trnF region. Our results strongly
support classifying Thottea s.l. within the subfamily
Aristolochioideae.
Aristolochia Sensu Lato. Our analyses of three dif-
ferent genes from the chloroplast and nuclear genomes
provide a more detailed phylogeny of Aristolochia s.l.
than do the studies of Murata et al. (2001) or Neinhuis
et al. (2005). All trees obtained indicate that Aristolochia
s.l. is a monophyletic group, consisting of two major
lineages (Figs. 2, 3). These lineages are congruent with
the framework of the subtribes Aristolochiinae and Is-
otrematinae established by Huber (1993). Furthermore,
our phylogenies based on three genes indicated that
the Isotrematinae clade is subdivided into two distinct
subgroups (Figs. 2, 3), whereas only the matK phylog-
eny suggests that the Aristolochiinae clade may also
be divided into two subgroups (Fig. 3). The matK phy-
logeny is more congruent with the framework consist-
ing two major groups and four subgroups proposed
by González and Stevenson (2002), with Aristolochia
s.s. and Pararistolochia in Aristolochiinae and Endodeca
and Isotrema in Isotrematinae, than with the classifi-
cation in which Aristolochia s.l. is deemed to be one
large genus with three subgenera (Schmidt 1935; Ma
1989; González 1999; González and Stevenson 2000).
2006] OHI-TOMA ET AL.: ARISTOLOCHIA MOLECULAR PHYLOGENY 489

FIG. 3. Continued.
490 SYSTEMATIC BOTANY
All phylogenies support the distinction of Endodeca
and Isotrema (Figs. 2, 3). The reciprocal monophyly of
the Aristolochia s.s. clade and the Pararistolochia clade
only appears in the matK phylogeny (Fig. 3), and is not
observed in the rbcL or phyA phylogeny (Fig. 2). There-
fore, we suggest that Pararistolochia should be treated
below the rank of genus. Chromosome number should
also be considered together with morphology in any
revision of the taxonomic ranks of the clades, although
González and Stevenson (2002) did not emphasize
chromosome number in their treatment of Aristolochia
s.l. Because the Isotrematinae clade, consisting of Iso-
trema and Endodeca, is characterized by the chromo-
some number 2n ! 32, we suggest that the clade
should be treated as a genus. In conclusion, based on
molecular phylogeny and chromosome numbers, as
well as on morphology, we provisionally propose a re-
vised classification of Aristolochia s.l. that divides the
genus into two genera with four subgenera: genus
Aristolochia including subgenera Aristolochia and Par-
aristolochia, and genus Isotrema including subgenera Is-
otrema and Endodeca. In the following sections, we sum-
marize the characteristics of each group of our pro-
posed system.
Aristolochia. The group is characterized by more
than three lobes of the gynostemium, a solitary anther
on the outer surface of each gynostemium lobe, a peri-
anth with one to three lobes, and dehiscent capsules
or fleshy indehiscent fruit (Huber 1993; González and
Stevenson 2002). The group corresponds to the sub-
tribe Aristolochiinae (Huber 1993) or Aristolochia sub-
genera Aristolochia and Pararistolochia (González 1999).
The group includes two subgroups (Fig. 3), and we
propose that they are recognized as the subgenera
Aristolochia and Pararistolochia. Subgenus Aristolochia is
characterized by a dehiscent capsule, lobes of the peri-
anth unilaterally appressed in the bud and breaking
up into one to three segments, six or fewer lobes of
the gynostemium, and six or fewer anthers. The spe-
cies of this group are widely distributed (Fig. 3). They
are morphologically quite diverse and several lower
taxonomic ranks may be recognized (Schmidt 1935;
González 1999). However, contrary to González and
Stevenson (2002), our matK phylogeny did not indicate
reciprocal monophyly of sections Gymnolobus and Di-
plolobus, or series Hexandrae and Thyrsicae of the former
section (Fig. 3). The main incongruence between the
morphological classification and the molecular phylog-
eny is found in the position of A. burelae and A. gran-
diflora. Subgenus Aristolochia also exhibits several chro-
mosome numbers. In our study, four different num-
bers, 2n ! 6, 12, 14, and 16, were observed and are
congruent with the phylogeny (Figs. 1, 3). Our study
did not include most species from the Mediterranean
region, which possibly belong to subgenus Aristolochia,
[Volume 31
and their chromosome numbers are more diverse, 2n
! 8, 10, 24, and 36 (Nardi 1984).
Subgenus Pararistolochia has fleshy indehiscent
fruits, a three-lobed perianth, valvate in bud, some-
times with one or three long tails, six to 12 lobes of
the gynostemium, six to 24 anthers, and chromosome
number of 2n ! 12 (Fig. 1; Table 1). About 33 species
in the Southern Hemisphere are recognized, one in Ka-
limantan, 22 in Papua New Guinea and northeast Aus-
tralia, and about 10 in western tropical Africa (Hutch-
inson and Dalziel 1954; Parsons 1996; González 1999;
González and Stevenson 2002). The hypothesis that
subgenus Pararistolochia is the most primitive group in
Aristolochia s.l. because of its morphological similarity
to Thottea s.l., remarked by Ma (1989), is not supported
by the molecular phylogeny. The subgenus has larger
chromosomes than other subgenera (Fig. 1). Further-
more, there are size differences in the chromosomes
among the African and Australian species (Fig. 1). In
the subgenus, three sections were recognized by Ma
(1992), Pararistolochia, Pararistolochioides, and Aristolo-
chioides. Because we examined only three species, two
from Africa and one from Australia, and did not sam-
ple A. decandra from the Malaysian region, a more de-
tailed examination is required in order to clarify the
historical biogeography of the group in the Southern
Hemisphere.
Isotrema. This group, comprising more than 50
species, has a gynostemium with three lobes, anthers
paired on the outer surface of each gynostemium seg-
ment, a perianth with three lobes, valvate in bud, and
an apically dehiscent capsule. The group is identical to
the subtribes Isotrematinae sensu Huber (1993) or to
Aristolochia subgenus Siphisia (González 1999). The
group has a higher chromosome number (2n ! 32)
than Aristolochia s.s., and the phyA phylogeny suggests
a gene duplication by polyploidization in the ancestor
of the genus (Fig. 2B). The group includes two sub-
groups, and we propose that they are recognized as
subgenera Isotrema and Endodeca (Fig. 3A). For Endo-
deca, this will necessarily involve a change in taxonom-
ic status. Subgenus Isotrema, which includes most spe-
cies of the genus occurring in North and Central
America and in East Asia, are erect herbs, herbaceous
vines, woody liana vines, or shrubs. The phylogeny
does not correspond completely to the species distri-
bution areas. Subgenus Endodeca is a herbaceous group
in North America, with reduced subtending leaves,
clasping bracts, short internodes in the laterals, and
prostrate inflorescence (Huber 1993; González and Ste-
venson 2002).
ACKNOWLEDGEMENTS. We thank the Botanical Garden, Bonn,
Germany, the Royal Botanic Garden Edinburgh, Scotland, and the
Kochi Pref. Makino Botanical Garden, Japan for providing sam-
ples, and Dr. D. Boufford for reading the manuscript. This study
was supported by Grants-in-Aid from the Japanese Ministry of
2006] OHI-TOMA ET AL.: ARISTOLOCHIA MOLECULAR PHYLOGENY
Education, Science and Culture (Nos. 12440240, and No.1 3375003,
17255004 to J. Murata).
LITERATURE CITED
APG (ANGIOSPERM PHYLOGENY GROUP). 2003. An update of the
angiosperm phylogeny group classification for the orders and
families of flowering plants: APG II. Botanical Journal of Lin-
nean Society 141: 399–436.
BORSCH, T., K. W. HILU, D. QUANDT, V. WILDE, C. NEINHUIS, and
W. BARTHLOTT. 2003. Noncoding plastid trnL-trnF sequences
reveal a well resolved phylogeny of basal angiosperms. Jour-
nal of Evolutional Biology 16: 558–576.
DOYLE, J. J. and J. L. DOYLE. 1987. A rapid DNA isolation proce-
dure for small quantities of fresh leaf tissue. Phytochemical
Bulletin 9: 11–15.
FAN, C. and Q.-Y. XIANG. 2003. Phylogenetic analyses of Cornales
based on 26S rRNA and combined 26S rDNA-matK-rbcL se-
quence data. American Journal of Botany 90: 1357–1372.
GOLDBLATT, P and D. E. JOHNSON. 1991. Index to plant chromo-
some numbers for 1988–1989. Monographs in Systematic Botany
from the Missouri Botanical Garden 30: 1–243.
GONZÁLEZ, F. 1999. Inflorescence morphology and the systematics
of Aristolochiaceae. Systematics and Geography of Plants 68:
159–172.
——— and D. W. STEVENSON. 2000. Perianth development and sys-
tematics of Aristolochia. Flora 195: 370–391.
——— and ———. 2002. A phylogenetic analysis of the subfamily
Aristolochioideae (Aristolochiaceae). Revista de la Aacademia
Colombiana de Ciencas Exactas, Fı́sicas y Naturales 26: 25–60.
GREGORY, M. P. 1956. A phyletic rearrangement in the Aristolo-
chiaceae. American Journal of Botany 43: 110–122.
HASEGAWA, M., H. KISHINO, and T. YANO. 1985. Dating of the
human-ape splitting by a molecular clock of mitochondrial
DNA. Journal of Molecular Evolution 22: 160–174.
HUBER, H. 1993. Aristolochiaceae. Pp. 129–137 in The families and
genera of vascular plants, vol. 2, eds. K. Kubitzki, J. G. Rohwer,
and V. Bittrich. Berlin: Springer.
HUTCHINSON, J. and J. M. DALZIEL. 1954. Aristolochiaceae. Pp. 77–
81 in Flora of West Tropical Africa. Ed. 2, vol. 1 (1). London:
Crown Agents.
JARAMILLO, M. A. and E. M. KRAMER. 2004. APETALA3 and PIS-
TILLATA homologs exhibit novel expression patterns in the
unique perianth of Aristolochia (Aristolochiaceae). Evolution
and Development 6: 449–458.
———, P. S. MANOS, and E. A. ZIMMER. 2004. Phylogenetic rela-
tionships of the perianthless Piperales: reconstructing the
evolution of floral development. International Journal of Plant
Sciences 165: 403–416.
KELLY, L. M. and F. GONZÁLEZ. 2003. Phylogenetic relationships
in Aristolochiaceae. Systematic Botany 28: 236–249.
KLOTZSCH, F. 1859. Die Aristolochiaceae des Berliner Herbariums.
Monatsberichte der Königlich Preussischen Akademie der Wissen-
schaften zu Berlin 571–625.
MA, J. S. 1989. A revision of Aristolochia Linn. from E. & S. ASIA.
Acta Phytotaxonomica Sinica 27: 321–364.
———. 1992. A taxonomic revision on genus Aristolochia subgenus
Pararistolochia. Acta Phytotaxonomica Sinica 30: 508–514.
MATHEWS, S. and M. J. DONOGHUE. 1999. The root of angiosperm
phylogeny inferred from duplicate phytochrome genes. Sci-
ence 286: 947–950.
——— and ———. 2000. Basal angiosperm phylogeny inferred
from duplicate phytochrome A and C. International Journal of
Plant Sciences 161: S41–S55
MURATA, J., T. OHI, S. WU, D. DARNAEDI, T. SUGAWARA, T. NAK-
ANISHI, and H. MURATA. 2001. Molecular phylogeny of Aris-
tolochia (Aristolochiaceae) inferred from matK sequences. Acta
Phytotaxonomica et Geobotanica 52: 75–83.
491
NARDI, E. 1984. The genus Aristolochia L. (Aristolochiaceae) in Italy
Publication. Webbia 38: 221–300.
NEINHUIS, C., S. WANKE, K. W. HILU, K. MÜLLER, and T. BORSCH.
2005. Phylogeny of Aristolochiaceae based on parsimony,
likelihood, and bayesian analyses of trnL–trnF sequences.
Plant Systematics and Evolution 250: 7–26.
NICKRENT, D. L., A. BLARER, Y. L. QIU, D. E. SOLTIS, P. S. SOLTIS,
and M. ZANIS. 2002. Molecular data place Hydnoraceae with
Aristolochiaceae. American Journal of Botany 89: 1809–1817.
PARSONS, M. J. 1996. New species of Aristolochia and Pararistolochia
(Aristolochiaceae) from Australia and New Guinea. Botanical
Journal of Linnean Society 120: 199–238.
PONCY, O. 1978. Le genre Pararistolochia, Aristolochiaceae
D’Afrique tropicale. Adansonia 17: 465–494.
POSADA, D. and K. CRANDALL. 1998. Modeltest: testing the model
of DNA substitution. Bioinformatics 14: 817–818.
QIU, Y. L., M. W. CHASE, D. H. LES, and C. R. PARKS. 1993. Mo-
lecular phylogenetics of the Magnoliidae: cladistic analyses
of nuclear sequences of the plastid gene rbcL. Annals of the
Missouri Botanical Garden 80: 587–606.
———, J. LEE, F. BERNASCONI-QUADRONI, D. E. SOLTIS, P. S. SOLTIS,
M. ZANIS, E. A. ZIMMER, Z. CHEN, V. SAVOLAINEN, and M.
W. CHASE. 1999. The earliest angiosperms: evidence from mi-
tochondrial, plastid and nuclear genomes. Nature 402: 404–
407.
SAVOLAINEN, V., M. W. CHASE, S. B. HOOT, C. M. MORTON, D. E.
SOLTIS, C. BAYER, M. F. FAY, A. Y. DE BRUIJN, S. SULLIVAN,
and Y. L. QIU. 2000. Phylogenetics of flowering plants based
on combined analysis of plastid atpB and rbcL gene sequenc-
es. Systematic Biology 49: 306–362.
SCHMIDT, O. C. 1935. Aristolochiaceae. Pp. 204–242 in Die natür-
lichen Pflanzenfamilien. Ed. 2., vol.16b, eds. A. Engler and K.
Prantl. Lepipzig: Engelmann.
SOLTIS, D. E., P. S. SOLTIS, M. W. CHASE, M. E. MORT, D. C. AL-
BACH, M. ZANIS, V. SAVOLAINEN, W. H. HAHN, S. B. HOOT,
M. F. FAY, M. AXTELL, S. M. SWENSEN, L. M. PRINCE, W. J.
KRESS, K. C. NIXON, and J. S. FARRIS. 2000. Angiosperm phy-
logeny inferred from 18S rDNA, rbcL, and atpB sequences.
Botanical Journal of Linnean Society 133: 381–461.
STELLARI, G. M., M. A. JARAMILLO, and E. M. KRAMER. Evolution
of the APETALA3 and PISTILLATA lineages of MADS-Box
containing genes in the basal angiosperms. Molecular Biology
and Evolution 21: 506–519.
SUGAWARA, T, J. MURATA, S. WU, T. OHI, T. NAKANISHI, and H.
MURATA. 2001. A cytological analysis of 24 taxa in Aristolo-
chia subgenera Siphisia and Aristolochia (Aristolochiaceae).
Acta Phytotaxonomica et Geobotanica 52: 149–158.
SWOFFORD, D. L. 2002. PAUP*. Phylogenetic analysis using parsi-
mony and distance (* and other methods). Version 4. Sun-
derland: Sinauer Associates.
THOMPSON, J. D., T. J. GIBSON, F. PLEWNIAK, F. JEANMOUGIN, and
D. G. HIGGINS. 1997. The CLUSTAL X windows interface:
flexible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Research 25: 4876–4882.
YU, W., L.-Q. HUANG, and C.-Y. CHENG. 1995. A preliminary cy-
totaxonomical study on the Chinese Aristolochia (Aristolochi-
aceae). Bulletin of Botanical Research 15: 199–205.
ZANIS, M. J., P. S. SOLTIS, Y. L. QIU, E. ZIMMER, and D. E. SOLTIS.
2003. Phylogenetic analyses and perianth evolution in basal
angiosperms. Annals of the Missouri Botanical Garden 90: 129–
150.
APPENDIX 1. List of taxa used in phylogenetic analyses, with
voucher information, and DDBJ/GenBank accession numbers. The
generic status is regarded as Aristolochia s.l.. Species are catego-
rized according to our proposed genera and subgenera, and are
listed alphabetically within each category.
Aristolochia L. subgenus Aristolochia: A. albida Duch., BG
 492 SYSTEMATIC BOTANY
Bonn 17419, Neinhuis 91 (BONN), rbcL AB205585, phyA AB206921,
matK AB211566. A. anguicida Jacq., Murata et al. JM03-140 (TI),
matK AB211575. A. arcuata Mast., Murata et al. SETS29 (TI), matK
AB060786. A. argentina Griseb., Murata et al. SETS33 (TI), matK
AB060787. A. baetica L., Murata et al. SETS5 (TI), matK AB060766.
A. brasiliensis Mart. & Zucc., Sugawara et al. SETS82 (TI), matK
AB071819. A. burelae Herzog, Murata et al. JM00-1 (TI), rbcL
AB205587, phyA AB206923, matK AB060788. A. cauliflora Ule,
Murata et al. SETS85 (TI), matK AB211559. A. clematitis L., Murata
et al. SETS17 (TI), rbcL AB205588, phyA AB206925, matK AB060767.
A. contorta Bunge, Murata et al. SETS95 (TI), matK AB211581. A.
cymbifera Mart. & Zucc., Murata et al. SETS55 (TI), matK
AB060789. A. debilis Sieb. & Zucc., Murata et al. SETS10 (TI), matK
AB060768. A. elegans Mast., Murata et al. SETS24 (TI), matK
AB060790. A. eriantha Mart. & Zucc., BG Bonn 12952, Neinhuis 99
(BONN), rbcL AB205590, phyA1 AB206928, phyA2 AB206929, matK
AB211564. A. esperanzae Kuntze, Murata et al. SETS88 (TI), matK
AB211573. A. fimbriata Cham. & Schltdl., Murata et al., SETS11
(TI), matK AB060791; Kuroiwa and Maeda 2301 (MBK), matK
AB211563. A. foveolata Merr. Murata et al. SETS4 (TI), matK
AB060769. A. gaudichaudii Duchtr., Murata et al. SETS8 (TI), matK
AB060775. A. gibertii Hook., Murata et al. SETS2 (TI), matK
AB0608792. A. gigantea Hook., Murata et al. SETS22 (TI), rbcL
AB205591, phyA AB206930, matK AB060793. A. goliathiana M.J.
Parsons, Murata et al. SETS19 (TI), matK AB060770. A. grandiflora
Sw., Sugawara et al. SETS75 (TI), rbcL AB205592, phyA AB206931,
matK AB071815. A. holostylis F. González, BG Bonn 02193, Nein-
huis 116 (BONN), rbcL AB205600, phyA1 AB206944, phyA2
AB206945, matK AB211568. A. hookeriana Craib, Murata et al.
JM00-2 (TI), matK AB060771. A. indica L., Murata et al. SETS93 (TI),
matK AB211579. A. jackii Steud., Murata et al. SETS56 (TI), matK
AB060776. A. kankauensis Sasaki, Murata et al. SETS35 (TI), matK
AB060777. A. kerrii Craib, Murata et al. JM02-260 (TI), matK
AB211572. A. littoralis Parodi, Murata et al. JM04-45 (TI), matK
AB211571. A. macroura Duchtr., Murata et al. SETS23 (TI), matK
AB060795. A. maxima L., Murata et al. SETS37 (TI), rbcL AB205594,
phyA AB206934, matK AB060783; Murata et al. JM01-1 (TI), matK
AB060782. A. micrantha Duchartre, Sugawara et al. SETS78 (TI),
rbcL AB205595, phyA1 AB206935, phyA2 AB206936, matK
AB071815. A. nelsonii Eastwood, Murata et al. JM98-212 (TI), matK
AB060781. A. odoratissima L., Murata et al. SETS31, matK
AB060798; RBGE 19592686, Ohi-Toma s.n. (TI), matK AB211561; Ta-
teishi and Takayama s.n. (TI), matK AB211585. A. ovalifolia Duchtr.,
Murata et al. SETS66 (TI), matK AB060785. A. papillifolia Ding
Hou, Murata et al. JM00-4 (TI), matK AB060772. A. paulistana
Hoehne, Murata et al. SETS91 (TI), matK AB211577. A. petersiana
Klotzsch, Murata et al. SETS89 (TI), matK AB211574. A. pentandra
Jacq., Neinhuis 106 (BONN), rbcL AB205596, phyA1 AB206941,
phyA2 AB206942, matK AB211565. A. peruviana O.C.Schimidt,
Murata et al. SETS70 (TI), matK AB060799. A. pierrei Lecomte, Mur-
ata et al. SETS64 (TI), matK AB060778. A. pilosa Kunth, Murata et
al. SETS6 (TI), matK AB060802. A. ringens Vahl, Murata et al.
SETS13 (TI), matK AB060800; Murata et al. SETS54 (TI), matK
AB060797. A. sempervirens L., McPherson s.n. RBGE19821706, Ohi-
Toma s.n. (TI), matK AB211560. A. tagala Cham., Murata et al.
SETS27 (TI), matK AB060779; Murata et al. SETS48 (TI), matK
AB060780; Ohi-Toma and Sugawara 040521 (TI), matK AB211567. A.
taliscana Hook.&Arn., Sugawara et al. SETS79 (TI), matK
AB071816. A. tentaculata O.C.Schmidt, Kajita et al. s.n. (TI), matK
AB211570. A. triangularis Cham., Murata et al. SETS20 (TI), matK
AB060803. A. trilobata L., Murata et al. STES1 (TI), matK
AB060804, A. zollingeriana Miq., Sugawara et al. SETS80 (TI), rbcL
AB205599, phyA AB206943, matK AB071817. A. aff. ringens, Kuroi-
wa and Maeda 2283 (MBK), matK AB211584; Kuroiwa and Maeda 2308
(MBK), matK AB211583; Kuroiwa and Maeda 588 (MBK), matK
View publication stats
[Volume 31
AB211586. Aristolochia spp. (unidentified species), JM01 from
China, Murata et al. SETS16 (TI), matK AB060773; JM02 from India,
Murata et al. SETS49 (TI), matK AB060774; JM03 from Australia,
Sugawara et al. SETS81 (TI), matK AB071818; JM04 from Myanmar,
Murata et al. s.n. (TI) matK AB211578; JM05 from Brazil, Murata et
al. 03-166 (TI), matK AB211580; JM06 from Brazil, Murata et al.
JM04-46 (TI), matK AB211576.
Aristolochia L. subgenus Pararistolochia (Hutchinson & Dal-
ziel) O.C.Schmidt: A. praevenosa F. Muell, Koishikawa BG, Ohi-
Toma, s.n. (TI) rbcL AB205601, phyA AB206946, matK AB211589. A.
promissa (Mast.) Keay, BG Bonn 13014, Neinhuis 118 (BONN), rbcL
AB205602, phyA AB206947, matK AB211690. A. triactina Hook.f.,
BG Bonn 12767, Neinhuis 119 (BONN), rbcL AB205603, phyA
AB206948, matK AB211588.
Isotrema Raf. subgenus Isotrema: A. arborea Linden, Murata et
al. JM99-1 (TI), rbcL AB205586, phyA1 AB206922, phyA2 AB206923,
matK AB060739. A. austroyunnanensis S.M. Hwang, Murata et al.
SETS15 (TI), matK AB060740. A. californica Torr., Sugawara et al.
SETS76 (TI), matK AB071813. A. cucurbitifolia Hayata, Murata et
al. SETS39 (TI), matK AB060741. A. kaempferi Willd., Murata et al.
SETS12 (TI), rbcL AB205593, phyA1 AB206932, phyA2 AB206933,
matK AB060743. A. kwangsiensis C.F. Liang, Murata et al. SETS28
(TI), matK AB060744. A. liukiuensis Hatus., Murata et al. SETS26
(TI), matK AB060745; Murata et al. SETS40 (TI), matK AB060746. A.
macrophylla Lam., Murata et al. SETS3 (TI), matK AB060742; data
from GenBank, rbcL L12630. A. manshuriensis Kom., Murata et al.
SETS21 (TI), matK AB060748. A. mollissima Hance, Murata et al.
SETS30 (TI), matK AB060749. A. moupinensis Franch., Murata et al.
SETS9 (TI), matK AB060751; Murata et al. 2003337 (TI), matK
AB211562. A. nakaoi F. Maekawa, Murata et al. SETS14 (TI), matK
AB060752. A. neolongifolia J.K. Wu & Z.L.Yang, Murata et al.
SETS52 (TI), matK AB060750; Murata et al. SETS96 (TI), matK
AB211582. A. paracleta Pfeifer, Murata et al. JM97-206 (TI), matK
AB060758. A. saccata Hook.f. & Thoms., Murata et al. SETS86 (TI),
matK AB211569. A. salvadorensis Standl., Murata et al. JM00-3 (TI),
matK AB060759. A. shimadai Hayata, Murata et al. SETS7 (TI), matK
AB060756; Murata et al. SETS38 (TI), matK AB060760; Murata et al.
SETS43 (TI), matK AB060753; Murata et al. SETS51 (TI), matK
AB060762; A. tomentosa Sims, Sugawara et al. SETS77 (TI), rbcL
AB205598, phyA1 AB206937, phyA2 AB206938, matK AB071814. A.
tricaudata Lem., Murata et al. SETS42 (TI), matK AB060764. A.
wardiana J.S. Ma, Murata et al. SETS98 (TI), matK AB211556. A.
westlandii Hemsl., Murata et al. SETS36 (TI), matK AB060765. A.
aff. kwangsiensis, Murata and Ohi-Toma V041219-40 (TI), matK
AB211587.
Isotrema Raf. subgenus Endodeca: A. convolvulacea Small, Mur-
ata et al. SETS83 (TI), rbcL AB205589, phyA1 AB206927, phyA2
AB206928, matK AB211557. A. serpentaria L., Murata et al. SETS84
(TI), rbcL AB205597, phyA1 AB206939, phyA2 AB206940, matK
AB211558.
ARISTOLOCHIACEAE, subfamily ARISTOLOCHIOIDEAE:
Thottea borneensis Valeton, Bogor BG of Indonesia XIBXIII134,
Ohi-Toma s.n. (TI), rbcL AB205604, matK AB211554. T. macrantha
(Boerl.) Ding Hou, Bogor BG of Indonesia XIBXIII133, Ohi-Toma
s.n. (TI), rbcL AB205605, matK AB211555. T. tomentosa (BI.) Ding
Hou, Murata et al. JM01-2 (TI), rbcL AB205606, phyA AB206951,
matK AB060738.
ARISTOLOCHIACEAE, subfamily ASAROIDEAE: Asarum
canadense L., data from GenBank, rbcL L14290. A. caudigerum
Hance, Koishikawa B.G. Ohi-Toma s.n. (TI), rbcL AB205608, matK
AB211553. A. yakusimense Masam., Koishikawa B.G. Murata et al.
s.n. (TI), rbcL AB205609, phyA AB206949, matK AB060737. Saruma
henryi Oliver, Murata et al. SETS36 (TI), rbcL AB205607, phyA
AB206950, matK AB060736.
LACTORIDACEAE: Lactoris fernandeziana Phil., data from
GenBank, rbcL L08763, phyA AF190091, matK AF543739.