REGULAR PAPER

Morphology and phylogenetics of two holoparasitic plants,Balanophora japonica and Balanophora yakushimensis(Balanophoraceae), and their hosts in Taiwan and Japan

Huei-Jiun Su • Jin Murata • Jer-Ming Hu

Received: 14 March 2011 / Accepted: 2 August 2011 / Published online: 6 September 2011

� The Botanical Society of Japan and Springer 2011

Abstract Balanophora japonica and B. yakushimensis

are two putatively agamospermic taxa previously reported

from southern Japan. Their inflorescences superficially

represent those of B. laxiflora and B. fungosa. In this study

we confirmed their presence in Taiwan by morphological

and phylogenetic analysis using nuclear 18S rDNA and

nrITS sequences with related taxa. B. japonica, B. yaku-

shimensis, and B. laxiflora formed a well-supported clade

that is distinct from other Balanophora. All three taxa also

show considerable differences on morphological and

nucleotide sequence differences, therefore the name of

B. yakushimensis is retained. The results provide new

insights on the intrageneric classification of Balanophora

and suggest the positioning of female flowers should be

down-weighted. We also successfully identify the hosts of

B. japonica and B. yakushimensis by amplifying chloro-

plast matK sequences from the connected root tissues. The

results showed that B. japonica parasitizes on Symplocos

species, and that B. yakushimensis parasitizes on Distylium

racemosum in Japan and Schima superba in Taiwan’s

population.

Keywords Balanophora � Balanophoraceae �Agamospermic � Phylogeny � Host plant � Parasitic plant

Introduction

Balanophora (Balanophoraceae) is mainly distributed in

temperate and tropical Asia, and extends to the areas of

tropical Africa, Madagascar and tropical Australia (Hansen

1972). Balanophora species are either dioecious or

monoecious and are holoparasites on several groups of

dicotyledonous trees. Holoparasitic plants are heterotrophic

and can serve as excellent models for understanding the

evolution of photosynthesis and chloroplasts in plants

(Nickrent et al. 1997; Barbrook et al. 2006). Most of the

holoparasites have been found to retain a small plastid

genome with size of 43 kb (Conopholis) to 72 kb (Epifa-

gus), however Balanophora is one of the very few holo-

parasitic taxa for which no plastome sequence has yet to be

identified (Nickrent et al. 1997; Nickrent and Garcia 2009).

In addition, Balanophora, similar to Rafflesia (Rafflesia-

ceae), is an example of holoparasite that shows some of the

most divergent nuclear 18S ribosomal DNA sequences in

flowering plants (Nickrent and Starr 1994; Nickrent et al.

2004), demonstrating the extremes of plant genome

evolution.

The morphology of Balanophora species is highly

reduced. The male flowers of Balanophora have one whorl

of perianth and the anthers always form a synandrium. The

female inflorescences contain thousands of tiny flowers,

each having only an ovary that is situated by a sterile, club-

shaped structure called a spadicle (Hansen 1972) or clav-

iform body (Eberwein et al. 2009). The spadicle is usually

larger than the ovary and all the spadicles from the many

flowers form the surface of the female inflorescences.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10265-011-0447-5) contains supplementarymaterial, which is available to authorized users.

H.-J. Su � J.-M. Hu (&)

Institute of Ecology and Evolutionary Biology, National Taiwan

University, Rm 1227, Life Science Building, 1 Sec. 4,

Roosevelt Road, Taipei 106, Taiwan

e-mail: jmhu@ntu.edu.tw

J. Murata

Botanical Gardens, Graduate School of Science,

University of Tokyo, Tokyo, Japan

123

J Plant Res (2012) 125:317–326

DOI 10.1007/s10265-011-0447-5

Hansen (1972, 1999) recognized 15 species in two

subgenera of Balanophora: subgen. Balanophora and

subgen. Balania. In subgen. Balanophora the male flowers

are 4–6-merous with triporate or polyporate pollen and the

female flowers are situated both on the main inflorescence

axis and/or lower part of spadicles, whereas in subgen.

Balania the male flowers are 3-merous with inaperturate

pollen grain and the female flowers are only situated on the

inflorescence axis. However, this classification was ques-

tioned by Murata (1990) who stated that the agamospermic

Balanophora species in Japan are morphologically similar

to each other, and cannot be readily placed in either of

Hansen (1972)’s proposed subgenera.

Four species were included in subgen. Balania by

Hansen (1972): Balanophora involucrata Hook. f.,

Balanophora wrightii Mak. (=Balanophora tobiracola

Mak.), Balanophora harlandii Hook. f., and Balanophora

japonica Mak. Balanophora japonica has only female

individuals known from Japan and has been determined to

be agamospermic (Kuwada 1928; Hansen 1972). There are

two other potentially agamospermic taxa (subgen. Balan-

ophora) noted by Hansen (1972), Balanophora fungosa

ssp. indica var. globosa (Jungh.) B. Hansen and Balano-

phora elongata Bl., both of which are restricted to Java

and/or Sumatra (Fagerlind 1948; Hansen 1972; Sunaryo

1992).

In contrast to Hansen (1972)’s treatment, Watanabe and

Akuzawa (1982) and Murata (1990) both distinguished

Balanophora nipponica Mak., a taxon including Balano-

phora kiusiana Ohwi (Murata 1992), from B. japonica and

showed that the former has labyrinth-like cuticular ridges

on the surface of the spadicles, whereas B. japonica has

short ridges similar to Balanophora laxiflora Hemsl. In

addition, the inflorescence of B. nipponica is pale orange

to reddish, versus being red in B. japonica in the field.

Balanophora nipponica is known only from Honshu,

Shikoku, and Kyushu in Japan (Murata 1990).

Another agamospermic species, Balanophora yaku-

shimensis Hatusima et Masamune, was first described by

Hatushima (1971) and was neglected in Hansen (1972)’s

revision, but later was treated as a synonym of B. fungosa

spp. indica (Arn.) Hansen (Hansen 1999). However, most

Japanese botanists keep it as a distinct species (Watanabe

and Akuzawa 1982; Murata in Yahara et al. 1986; Murata

1990; Minamitani 2009). This species was originally

known only from forests around altitude 600–900 m of

Yakushima Island, Japan. Later it was also found on

Amamioshima Island and Takakumayama in southern

Kyushu (Watanabe and Akuzawa 1982; Murata in Yahara

et al. 1986), and Murata (1990) further included one

specimen (Ohashi et al. 12827) collected from southern

Taiwan under the name of B. yakushimensis. Balanophora

yakushimensis is similar to B. japonica and B. nipponica in

general appearance, but the colors of the female flowers

(ovaries) are reddish in B. yakushimensis versus being

yellow in B. japonica and B. nipponica.

The highly reduced morphology of holoparasitic plants

has complicated their taxonomic classification, but the

utilization of molecular markers in recent years has helped

to address this issue. For example, Balanophoraceae is

sometimes associated with Rafflesiales, Hydnoraceae, and

Santalales; but phylogenetic analysis of nuclear 18s rDNA

and mitochondrial matR sequences clearly demonstrated

that Rafflesiales and Hydnoraceae are both distantly related

to Santalales, where Balanophoraceae showed a close

relationship to it (Nickrent et al. 2005). At a lower taxo-

nomic level, delimitation of Balanophora species is diffi-

cult because of their highly reduced morphological

features. Agamospermic taxa present even a greater chal-

lenge because most of the morphological characters typi-

cally analyzed in this family are those of male flowers, but

in agamospermic species only female plants are available.

In addition, phylogenetic analysis for the species of

Balanophora and their hosts has yet to be carried out with

molecular markers. For Balanophora, one of the challenges

is the difficulty of DNA extraction since it usually contains

a high amount of polysaccharides that can interfere with

PCR amplification. Another difficulty is the high nucleo-

tide substitution rate in Balanophoraceae (Nickrent and

Duff 1996) causing universal primers to not work well.

However, the high substitution rates of nuclear 18S ribo-

somal DNA sequences in Balanophora (Nickrent and Starr

1994; Nickrent and Duff 1996) actually provide a unique

opportunity to use them in inter- and intra-generic phylo-

genetic analysis within Balanophoraceae, and here we

provide the evidence that they are well suited for recon-

structing Balanophora species phylogeny.

Balanophora species are known to parasitize on a wide

range of host plants, with 70 host species from 35 families

had been reported (Hansen 1972). The most widely dis-

tributed species B. fungosa is reported to parasitize on

more than 24 hosts species from 15 families, and

B. japonica parasitizes 15 hosts from six families. Hansen

(1972) therefore considered there to be no correlation

between Balanophora species and their host plants. How-

ever, it is always difficult to accurately identify the host

species since the host roots are always underground and the

host’s trunk can be far from the haustoria. Recently, host

identification from root fragments has been successful by

PCR amplification of plastid DNA (Linder et al. 2000;

Brunner et al. 2001; Frank et al. 2010). The results can be

compared with NCBI GenBank for at least genus-level

identification of the host.

During the survey of Balanophora taxa in Taiwan, we

encountered five populations of Balanophora that were

putatively agamospermic since only female individuals can

318 J Plant Res (2012) 125:317–326

123

be found locally. After careful examination, three of them

are morphologically similar to B. yakushimensis and the

other two are similar to B. japonica. In the present study,

we collected samples of related taxa from Taiwan and

southern Kyushu, including Yakushima Island, and con-

firmed the presence of B. yakushimensis and B. japonica in

Taiwan based on morphological and molecular data. Floral

morphology was examined under light microscope and

scanning electron microscope. Phylogenetic analyses for

selected Balanophora species were conducted using

nuclear 18s ribosomal DNA and ITS sequences. Host plant

identification was performed by identifying chloroplast

matK sequences determined by PCR amplification of root

materials.

Materials and methods

Plant materials

We collected Balanophora tissue samples from field pop-

ulations in Taiwan and four different sites from Yakushima

Island and Kagoshima, Kyushu, Japan, as well as from an

herbarium sample from Yunnan, China (see S1 for voucher

information). Samples containing the attached host plant

roots (if any) were carefully washed and preserved for

DNA analysis. A total of 22 host roots were collected from

the tissues connected to either B. japonica or B. yaku-

shimensis. The identification of plant species in the vicinity

of the Balanophora collections were recorded for refer-

ences. Total genomic DNAs were extracted from 0.1 g of

Balanophora inflorescences and host plant roots separately

according to standard CTAB extraction methods (Doyle

and Doyle 1987). The DNA of the herbarium specimen was

extracted using a Tri-Plant Genomic DNA reagent kit

(Geneaid Biotech Ltd., Taipei, Taiwan).

DNA sequencing and phylogenetic analysis

In this study, the nuclear ribosomal 18S (nr18S) and nr ITS

sequences of Balanophora species and the chloroplast

matK regions of host plant species were determined by

PCR and the subsequent sequencing. PCR reactions were

conducted with Blend Taq Plus polymerase (Toyobo,

Osaka, Japan) and reagent conditions according to the

manufacturer’s instructions. The primers used in this study

are listed in supplementary data (S2). The PCR specifica-

tion of nr18S regions was 94�C for 3 min, 35 cycles of

94�C for 50 s, 55�C for 50 s, 72�C for 1 min, and a final

extension of 72�C for 3 min. The PCR conditions for nrITS

were 94�C for 3 min, 35 cycles of 94�C for 30 s, 55�C for

40 s, 72�C for 45 s, and a final extension of 72�C for

3 min. Chloroplast matK amplification conditions were

similar to nrITS except for an annealing temperature at

52�C. All PCR products were then purified using a PCR

Purification kit (QIAGEN, Hilden, Germany) and both

strands of DNA were sequenced by ABI Prism 3700� DNA

Analyzer at the Institute of Plant and Microbial Biology

DNA analysis core laboratory, Academia Sinica, Taipei,

Taiwan.

Sequences of nr18S and nrITS were identified from

B. fungosa ssp. fungosa, B. tobiracola (previously named

B. wrightii), B. harlandii, B. laxiflora (two accessions), and

the individuals of B. japonica and B. yakushimensis in

Taiwan (two accessions each). Sequences of nrITS regions

were also identified from four other B. japonica and four

other B. yakushimensis individuals. Sequences obtained

directly from GenBank of nr18S of B. fungosa (L24044)

and eight other genera from Balanophoraceae (Nickrent

et al. 2005) were included in the analysis. One herbarium

sample of B. fungosa ssp. indica, collected by C. I. Peng

from Yunnan, China (Peng 18661) was used for the

amplification of nr18S/nrITS regions. The sequence of

Balanophora reflexa Becc. (EU598798) was omitted from

the full-length sequence analysis since it is too short and

the ITS1 region is too variable to obtain confident align-

ment with other Balanophora sequences. The data set for

phylogenetic reconstruction contains 17 sequences,

including those from nine taxa within Balanophora and

those from eight genera in the Balanophoraceae (see S1 for

voucher information). Regions with unavailable data, e.g.

nrITS of several Balanophoraceae genera, were treated as

missing data, a common approach in supertree/supermatrix

phylogenetic analysis (de Queiroz and Gatesy 2007). If

there are more than one identical sequences within a taxon,

only one of them were included in phylogenetic analyses to

reduce tree-searching time, i.e. the ones within B. japonica

(Su031, Su032, Hu1767, Hu1769, Hu1780, and Hu1805)

and B. yakushimensis (Su063, Su068, Su074, Hu1770,

Hu1779, and Hu1808).

The nrITS region of Balanophora species was initially

aligned with Clustal X 1.83 (Thompson et al. 1997) and

subsequently adjusted visually using MacClade 4.06

(Maddison and Maddison 2000). Phylogenetic analyses

were conducted using neighbor-joining (NJ), maximum

parsimony (MP) and maximum likelihood (ML) methods

using PAUP* 4.01b10 (Swofford 2002). Branch-and-bound

searches were conducted for the MP analysis and branch

support was evaluated by 1,000 pseudo-replicates of heu-

ristic search with tree bisection–reconnection (TBR)

branch swapping with steepest-descent option in effect.

The ML analysis employed the GTR?G model estimated

by Modeltest 3.7 (Posada and Buckley 2004), and a heu-

ristic search with 100 random addition replicates and TBR

branch swapping with steepest-descent option in effect. NJ

analysis was performed with the GTR?G model and

J Plant Res (2012) 125:317–326 319

123

bootstrap supports were estimated with 5,000 replicates.

Bayesian inference (BI) was employed under HKY85 by

using MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001).

The BI analyses were conducted with four chains of

Markov chain Monte Carlo simulation, sampling one tree

per 100 runs for 5,000,000 generations, and the HKY85

nucleotide substitution model with gamma distribution for

rate categories. The first 1,000 trees were discarded before

calculating the node probability.

Scanning electron microscopy

Inflorescences, bracts, and tubers of Balanophora samples

were cut into small pieces and preserved in 70% EtOH and

then dehydrated in steps; in 70% EtOH for 10 min, 85%

EtOH for 20 min, 95% EtOH for 20 min, and 100% EtOH

for 20 min twice. The dehydrated samples were stored in

100% EtOH and were critical-point dried and mounted on

aluminum stubs. The specimens were coated with gold and

observed with a Hitachi S-520 scanning electron

microscope.

Results

Morphological observations

Both B. japonica and B. yakushimensis are morphologi-

cally similar to and easily mistaken with the female

individuals of B. laxiflora. However, B. japonica and

B. yakushimensis can be distinguished from B. laxiflora by

comparison of ultra-structure characteristics (Fig. 1), and

nucleotide sequences, as revealed in this study. The mor-

phological features within species are consistent among

observed samples, more than 17 specimens were observed

for B. japonica and more than 20 specimens for B. yaku-

shimensis were observed (accessions given in S1). These

three taxa can be separated by their female flower posi-

tioning, the surface morphology of spadicles, and the color

of the ovaries. A comparison of the morphological char-

acters among the three taxa is provided in Table 1.

Although B. japonica and B. yakushimensis are super-

ficially similar to each other, they can be readily distin-

guished by several morphological features, as revealed by

previous studies. The major difference between the two

species is that female flowers of B. yakushimensis are

situated both in the lower parts of the spadicles and the

main axis of the inflorescence (Fig. 1f, g), whereas in

B. japonica they are usually only on the main inflorescence

axis (Fig. 1b, c). Similar to B. yakushimensis, B. laxiflora

also has female flowers in the lower parts of the spadicles

and the main axis of the inflorescence, but the flowers

are not as closely adnate to the spadicles as they are in

B. yakushimensis (Fig. 1f, g vs. j, k). In addition, the color

of the inflorescence axis and spadicles of B. japonica are

always red while the pistillate flowers (ovaries) are yellow

(Fig. 1a, b), similar to the ones of B. laxiflora (Fig. 1i, j). In

contrast, the inflorescence axis and spadicles of B. yaku-

shimensis are red to orange and the pistillate flowers

(ovaries) are red (Fig. 1e, f). The upper parts (the apex) of the

spadicles in all three species are obconical and the short

ridges on the surface of spadicles are visible, but incon-

spicuous on B. japonica and B. yakushimensis, compared to

B. laxiflora or B. fungosa ssp. fungosa (Fig. 1c, d, g, h, k–o).

Two other minor differences between B. japonica and

B. yakushimensis are seen on the bracts and tubers. The

margin of basal bracts is usually entire in B. japonica but

partially crenate or truncate in B. yakushimensis (S4 a, c) as

stated by Minamitani (2009). The cell shapes on the bracts

of the two species are similar under SEM, but the cells on

the margin of the bract apex are longer and flatter in

B. yakushimensis (S4 b, d).

Both of the tubers of B. japonica and B. yakushimensis

tubers are grouped in masses underground, but the tuber

shape and degree of branching are a little different. The

tubers of B. japonica are slightly elongated and the

branching point position is deeper than that of B. yaku-

shimensis. The tuber surface of B. japonica and B. yaku-

shimensis is composed of stellate wart cells and armature

cells, and the morphology and arrangement are quite sim-

ilar to each other.

Nucleotide diversity and phylogenetic relationships

The nr18S/nrITS data set contains 2,482 characters, 854 of

which are variable, and 533 of which are parsimony-

informative sites. The sequences were identical for speci-

mens from the same species for both B. japonica (six

accessions) and B. yakushimensis (six accessions), so only one

sequence from each of these species was included in phylo-

genetic analyses. There are 63 nucleotide substitution sites

(2.7% of p-distance) between the sequences of B. japonica

and B. yakushimensis, 12 of which are in the nr18S region and

the rest are in nrITS region.

Phylogenetic analyses from different methods revealed

the same tree topology. Only a single most parsimonious

tree was found in MP analysis with tree length = 1,539

(CI = 0.73, RI = 0.76). All Balanophora taxa form a

monophyletic group with solid support (100% MP, 91%

NJ, and 1.0 BI posterior probability) and sister to Langs-

dorffia hypogaea (Fig. 2). Two clades are found within

Balanophora, B. harlandii and B. tobiracola form the first

clade, and all other Balanophora formed a sister clade next

to it. Balanophora laxiflora, B. japonica, and B. yaku-

shimensis form a well-supported clade (100% MP, 100%

NJ, and 1.0 BI posterior probability), which is sister to the

320 J Plant Res (2012) 125:317–326

123

Fig. 1 Photos and scanning electron microscopy photographs of B.japonica, B. yakushimensis, B. laxiflora, and B. fungosa ssp. fungosa.

a–d B. japonica from Taiwan; a whole plant (Su077), b longitudinal

section of inflorescence showing female flowers (yellow ovaries)

clustered at the base of reddish spadicles (Su077), c SEM of

longitudinal section of inflorescence for better resolution on the

position of flowers (Su031), d epidermal surface at the top of the

spadicle (Su031); e–h B. yakushimensis from Taiwan; e whole plant

(Su083), f longitudinal section of inflorescence showing female

flowers (red ovaries) clustered at the base of reddish spadicles

(Su085), g SEM of longitudinal section of inflorescence for better

resolution on the position of flowers (Hsiao s. n.), h epidermal surface

at the top of the spadicle (Hsiao s. n.); i–l female plant of B. laxiflorafrom Taiwan; i whole plant (Su043), j longitudinal section of

inflorescence showing female flowers (yellow ovaries) clustered at the

base of reddish spadicles (Su080), k SEM of longitudinal section of

inflorescence for better resolution on the position of flowers (Su043),

l epidermal surface at the top of the spadicle (Su043); m epidermal

surface of spadicle of B. japonica from Kirishima, Japan (Murata s.

n.); n epidermal surface of spadicle of B. yakushimensis from

Amamioshima Isl., Japan (Murata 17106B); o epidermal surface of

spadicle of B. fungosa ssp. fungosa from Okinawa Isl. (Murata &

Nakajima 21468). Detailed voucher information is shown in S1. Barsb, f, j 0.5 mm; c, g, k 250 lm; d, h, l 50 lm; m, n, o 10 lm

J Plant Res (2012) 125:317–326 321

123

sequences of B. fungosa. Balanophora fungosa ssp. indica

is sister to the two B. fungosa ssp. fungosa sequences.

Balanophora japonica and B. yakushimensis are strongly

supported as being sister to each other (Fig. 2).

Host identification

All 22 host root samples of B. japonica and B. yakushim-

ensis were successfully identified by their chloroplast matK

sequences (Table 2). BLAST results allow these sequences

to be assigned at least to the genus level, and in some cases

they have perfect species matches (e.g. Distylium

racemosum). All of the hosts of B. japonica belong to

Symplocos spp. (Symplocaceae), either from Japan or

Taiwan, congruent with field observations. In contrast,

B. yakushimensis consistently parasitizes on D. racemosum

(Hamamelidaceae) in Japanese populations, and only

parasitizes to Schima superba var. superba (Theaceae) in

Taiwanese populations.

Discussion

In this study we described the morphological characteris-

tics of the two putatively agamospermic taxa B. japonica

and B. yakushimensis from Taiwan, and examined the

phylogenetic relationships of related taxa using ribosomal

DNA and the spacer sequences. The results clearly show

that B. yakushimensis is distinct from B. fungosa and

B. laxiflora, and can be readily separated from the agamo-

spermic taxon B. japonica.

The taxonomic classification of holoparasitic plants like

Balanophora has been very challenging, due to their

reduced morphological features that provide relatively little

information. In addition, these taxa usually show diverse

size and shape of inflorescences, particularly in the widely

distributed taxa like B. fungosa and B. laxiflora. Hansen

(1972, 1982, 1999) considered many of the morphological

differences present on specimens to only represent regional

variations within species and consequently reduced over a

hundred names to about 15 taxa in his treatments, mostly to

maintain taxonomic stability of species delimitation

(Hansen 1999). Although these arguments may hold true in

several occasions, local botanists usually prefer to recog-

nize those differences for convenience on field studies,

rather than to combine names, e.g. Hatushima (1971),

Watanabe and Akuzawa (1982), Huang and Murata (2003),

and Minamitani (2009).

We here employed molecular phylogenetics to facilitate

the delimitation of Balanophora in Taiwan and Japan,

particularly on the two agamospermic taxa, B. japonica and

B. yakushimensis and related taxa. The former was recog-

nized by Hansen (1972) and B. yakushimensis was treatedTa

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322 J Plant Res (2012) 125:317–326

123

under B. fungosa ssp. indica (Arn.) Hansen (Hansen 1999),

while these two taxa was considered to be different by

Murata (1990) based on spadicle morphology.

Hansen (1972) recognized the subgen. Balania of

Balanophora as having 3-merous male flowers with non-

aperturate pollen and female flowers not connate with the

spadicles. Subgen. Balania contains only four taxa:

B. involucrata, B. wrightii (=B. tobiracola), B. harlandii,

and B. japonica (Hansen 1972). The first three of these

species have rather easily recognizable male flowers, but

B. japonica is agamospermic and therefore lacks male

flower characters. The female-only nature of the agamo-

spermic taxa makes it extremely hard to place them in the

right intrageneric group, and Hansen (1972) eventually

emphasized on the position of the female flowers over

other characters for classification. It was at this point that

B. yakushimensis was placed under the names B. fungosa

ssp. indica because both of these taxa have female flowers

situated on the main inflorescence axis as well as at the

base of spadicles (Hansen 1999).

Three agamospermic species of Japanese Balanophora,

B. japonica, B. nipponica, and B. yakushimensis, were

recognized previously, based at least partially on differ-

ences in ultra-structures (Murata 1990). Balanophora nip-

ponica is distinguished from the other species by the

labyrinth-like cuticle surface of its spadicle. As mentioned

above, B. japonica and B. yakushimensis are very easy to

distinguish from each other based on the positions of the

female flowers (Murata 1990). In our observations it appears

that spadicle shape also are useful for calling into question the

placement of B. yakushimensis under B. fungosa ssp. indica,

with the spadicle of B. yakushimensis being shorter than the

typical B. fungosa ssp. indica spadicle. Additionally, the style

to ovary ratio (1–2 in B. yakushimensis) versus 4–5 in

B. fungosa ssp. indica (Hansen 1972), is useful in distin-

guishing these taxa.

There are three varieties of B. fungosa ssp. indica:

indica, minor, and globosa, where the later two taxa are

rare and only known in restricted areas (Hansen 1972). The

spadicle shape is considerably variable in B. fungosa ssp.

indica var. indica, the most widely distributed of the

varieties. It varies from being long obconical similar to

what is seen in B. fungosa ssp. fungosa, to obovoid, like in

other Balanophora (Hansen 1972). However, the conspic-

uous cuticular ridges on the surface of spadicles usually

formed a labyrinth-like pattern as in B. nipponica or

B. involucrata (Fig. 1; Hansen 1972), which is different

from B. yakushimensis (Fig. 1; Murata 1990).

Balanophora fungosa ssp. indica var. globosa is found

in Java and only female individuals have been observed. It

has a depressed inflorescence covered more than half by

the leaves/bracts (Hansen 1972), which is unique in the

genus. The tubers in B. fungosa ssp. indica var. minor are

elongated and cylindrical, different from other varieties of

Fig. 2 Phylogenetic

relationship of Balanophoraspecies and other taxa in

Balanophoraceae based on

nuclear 18S rDNA and nrITS

sequences. The phylogram is

based on the results of

maximum likelihood analysis.

Numbers above branches are

bootstrap supports from MP and

NJ analyses and numbers belowbranches are posterior

probabilities from BI analysis

J Plant Res (2012) 125:317–326 323

123

B. fungosa ssp. indica and B. yakushimensis. This variety

has been collected a few times in south India and once in

the mountains of northwest Thailand. Based on the above

morphological characters, B. yakushimensis is likely not

part of B. fungosa. This conclusion is further supported by

molecular data that shows B. yakushimensis as distinct

from all three B. fungosa sequences.

The two subspecies of B. fungosa, ssp. fungosa and ssp.

indica, are distinguished by their sexual expression, where

the former is monoecious and the latter is dioecious. In

addition, B. fungosa ssp. indica has a western distribution

from India to Indochina, while ssp. fungosa is found in East

Asia, including Taiwan and Ryukyu (Hansen 1972; Murata

1988). The B. fungosa ssp. fungosa sample in our study

was collected in southern Taiwan (Huang s.n.) with a

monoecious inflorescence. The B. fungosa ssp. indica

sample used here is from Yunnan, China (collection Peng

et al. 18661), and had a male inflorescence specimen,

therefore clearly belonging to B. fungosa ssp. indica var.

indica. The two subspecies of B. fungosa have very similar

floral morphology and they have been found occasionally

with both monoecious and dioecious individuals in the

same populations (Murata 1988), suggesting the delimita-

tion of the two subspecies is not absolute. The monophyly

of the three samples of B. fungosa in the molecular phy-

logeny indicates that the two subspecies are indeed closely

related, and is congruent with previous morphological

observations.

The results of phylogenetic analysis revealed a clear

topology within Balanophora, with B. laxiflora, B. japon-

ica, and B. yakushimensis forming a well-supported clade.

In this analysis, 2.7–3.3% of nucleotide sequence varia-

tions was phylogenetically informative for clearly distin-

guishing the three taxa. The fact that B. japonica is not

closely related to B. tobiracola (formerly B. wrightii) or

B. harlandii on the nrITS tree, suggests a re-consideration

of placing it under subgen. Balania. If B. japonica were

instead included in subgen. Balanophora, it and B. reflexa

would be the only taxa in the subgenus to have female

flowers situated only on main axis. This suggests that the

positions of female flowers should be down-weighted in

intrageneric classification. In any case, B. yakushimensis

should be treated separately from B. fungosa and its name

should be retained.

There is an nrITS sequence of B. reflexa (EU598798) on

GenBank, but the sequence was not alignable at the ITS1

region. Although we did not include it in the full-length

data set, we performed another phylogenetic analysis with

it by using only its 5.8S region and the nearby alignable

regions for the Balanophora species. The result showed

that it is distinct from all other Balanophora species (see

S3). Further phylogenetic analysis using 18S nrDNA

Table 2 List of Balanophora hosts identified by chloroplast matK sequences from the attached root tissues

Parasitic taxa Voucher no. Location Host root identified

B. japonica Hu1769 Yakushima, Japan Symplocos sp.

B. japonica Hu1780 Kirishima, Kagoshima, Japan Symplocos myrtacea

B. japonica Hu1786 Kirishima, Kagoshima, Japan Symplocos myrtacea

B. japonica Hu1826 Koba-dake, Kagoshima, Japan Symplocos sp.

B. japonica Su042 Mt. Syongkongnan, Taipei, Taiwan Symplocos sp.

B. japonica Su077 Mt. Syongkongnan, Taipei, Taiwan Symplocos sp.

B. japonica Su078 Mt. Syongkongnan, Taipei, Taiwan Symplocos sp.

B. japonica Su079 Mt. Syongkongnan, Taipei, Taiwan Symplocos sp.

B. japonica Hu1566 Mt. Daton, Taipei, Taiwan Symplocos sp.

B. japonica Hu1567 Mt. Daton, Taipei, Taiwan Symplocos sp.

B. japonica Hu1568 Mt. Daton, Taipei, Taiwan Symplocos sp.

B. yakushimensis Hu1771 Yakushima, Japan Distylium racemosum

B. yakushimensis Hu1779 Kirishima, Kagoshima, Japan Distylium racemosum

B. yakushimensis Hu1807 Koba-dake, Kagoshima, Japan Distylium racemosum

B. yakushimensis Hu1808 Koba-dake, Kagoshima, Japan Distylium racemosum

B. yakushimensis Su074-1 Mt. Shiaochu, Nantou, Taiwan Schima superba var. superba

B. yakushimensis Su074-2 Mt. Shiaochu, Nantou, Taiwan Schima superba var. superba

B. yakushimensis Su074-5 Mt. Shiaochu, Nantou, Taiwan Schima superba var. superba

B. yakushimensis Su083-1 Mt. Lala, Taoyuan, Taiwan Schima superba var. superba

B. yakushimensis Su083-2 Mt. Lala, Taoyuan, Taiwan Schima superba var. superba

B. yakushimensis Su083-3 Mt. Lala, Taoyuan, Taiwan Schima superba var. superba

B. yakushimensis Su083-4 Mt. Lala, Taoyuan, Taiwan Schima superba var. superba

324 J Plant Res (2012) 125:317–326

123

sequences on B. reflexa and other Balanophora will be

helpful since they already contain a fair amount of phylo-

genetic signals.

No intraspecific variation was found in our samples of

B. japonica and B. yakushimensis from Japan and Taiwan,

which is not surprising if they are indeed agamospermic.

We here provide evidences that the distribution of B.

japonica extends southward to northern Taiwan, and report

several new collections of B. yakushimensis from northern

and central Taiwan. In this study we found B. japonica on

Mt. Datong and Mt. Syongkongnan. B. laxiflora is also

found nearby on Mt. Syongkongnan. Both localities of B.

japonica are at 950 m altitude. Balanophora yakushimensis

was collected on Mt. Lala in the northern Taiwan and also

on Mt. Lucyu and Mt. Siaochu in central Taiwan, with all

collecting sites being at the elevation of 1,500–1,800 m.

Balanophora laxiflora appears to be the most common

taxon of Balanophora in Taiwan, and it is also distributed

from southern China to Vietnam. Taking into account

the close relationship of B. laxiflora, B. japonica, and

B. yakushimensis, and also the agamospermic nature of the

later two taxa, it is reasonable to assume the two agamo-

spermic species were derived from a bisexual ancestor like

B. laxiflora, and likely moved from south to north during

their evolution. The size and morphology of B. laxiflora

individuals, as well as their nrITS sequences, are quite

variable (Hu lab, field observations and unpublished data),

which would be congruent with this species being the

progenitor of B. japonica and B. yakushimensis.

The host identities were successfully revealed by

molecular markers in our survey by direct PCR of attached

host tissues. This is promising for the future accurate

identification of hosts and for making inferences about the

life history of the parasitic plants, since their presence

depends on the host’s distribution. Balanophora japonica

has been documented as a wide-host range taxon, parasit-

izing on more than 15 taxa from eight families (Hansen

1972), but mostly Symplocos species (Minamitani 2009;

Watanabe 1942). Our results showed all 11 host samples of

B. japonica to be indeed Symplocos species, but the exact

taxa are yet to be determined.

In contrast, the hosts of B. yakushimensis showed the

distinct pattern that it parasitizes D. racemosum in Japan

and S. superba in Taiwan. The former case was also sug-

gested by field observations in a previous study (Minami-

tani 2009). Distylium racemosum is distributed in southern

Japan (Kyushu), the Ryukyus, and is also found rarely in

southern Taiwan, China and Korea (Horikawa 1976), but it

never associates with Balanophora outside of Japan.

Schima superba, on the other hands, is found in South

China and Taiwan, but not in Japan. There is a subspecies

of Schima wallichii (DC.) Korth., S. wallichii ssp. liukiu-

ensis (Nak.) Bloemb., that is endemic to the Ryukyu

Islands, Japan (Horikawa 1972), but it has never been

reported to be associated with Balanophora. Interestingly,

S. wallichii has been reported as the host of another spe-

cies, B. fungosa (Hansen 1972). The associations of B.

yakushimensis and the two host plants suggest an inter-

esting pattern that may reflect ecological adaptation and

host switches during its expansion.

The hosts of B. laxiflora have yet to be determined by

molecular markers, but it is known that at least seven

species serve as its hosts and that none of these are species

of Symplocos or Schima (Hansen 1972). A thorough sam-

pling of B. laxiflora and related taxa will be necessary for

elucidating the evolutionary history of its host interactions.

Here we provide a key for Balanophora taxa in Taiwan

and Japan, based on female flower and inflorescence

morphology.

Key to female flowers/inflorescence of Balanophora

species in Taiwan and Japan

1. Spadicle apex smooth

2. Monoecious, male flowers intermingled with

female flowers .................................. B. tobiracola

2. Dioecious, male and female flowers on different

individuals........................................... B. harlandii

1. Spadicle apex with ridges on the cuticle surface

3. Plant monoecious*, male flowers positioned below

female ones; inflorescences yellow to orange-

yellow............................... B. fungosa ssp. fungosa

3. Plant dioecious or only female in population;

inflorescences red or orange-red

4. Female flowers both in the lower part of the

spadicles and on the main axis of the

inflorescences

5. Flowers (ovaries) yellow; dioecious

.................................................B. laxiflora

5. Flowers (ovaries) red; spadicle ridge

inconspicuous; agamospermic (females)

....................................... B. yakushimensis

4. Female flowers only on the main axis of the

inflorescences; agamospermic

6. Cuticular ridges on spadicle labyrinth

like ....................................... B. nipponica

6. Cuticular ridges on spadicle short

................................................ B. japonica

*Murata (1988) reported presence of unisexual (male)

individuals of B. fungosa ssp. fungosa, from Ishigaki

Island, the Ryukyus, Japan, but appeared to be quite rare.

J Plant Res (2012) 125:317–326 325

123

Acknowledgments We deeply appreciate Dr. Shu-Chuan Hsiao,

Mr. Tadashi Minamitani and Jiunn-Yih Huang for their help in

specimen collections. We also thank the technical assistance of

Technology Commons, College of Life Science, NTU with SEM, and

also the staffs at the herbaria of Harvard University (A, GH) and

Biodiversity Research Center, Academia Sinica (HAST) for the help

on literature survey and providing specimen samples. We thank David

W. Taylor for helps on editing the manuscript. This study was partly

supported by grants from National Science Council, Taiwan (NSC

96-2621-B-002-008-MY3 and 99-2918-I-002-020).

References

Barbrook AC, Howe CJ, Purton S (2006) Why are plastid genomes

retained in non-photosynthetic organisms? Trends Plant Sci

11:101–108

Brunner I, Brodbeck S, Buchler U, Sperisen C (2001) Molecular

identification of fine roots of trees from the Alps: reliable and

fast DNA extraction and PCR-RFLP analyses of plastid DNA.

Mol Ecol 10:2079–2087

de Queiroz A, Gatesy J (2007) The supermatrix approach to

systematics. Trends Ecol Evol 22:34–41

Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small

quantities of fresh leaf tissue. Phytochem Bull 19:11–15

Eberwein R, Nickrent DL, Weber A (2009) Development and

morphology of flowers and inflorescences in Balanophora pap-uana and B. elongata (Balanophoraceae). Am J Bot 96:1055–1067

Fagerlind F (1948) Bau und entwicklung der vegetativen organe von

Balanophora. Kungl Svenska Vetenskapsakademiens Handlingar

25:1–72

Frank DA, Pontes AW, Maine EM, Caruana J, Raina R, Raina S,

Fridley JD (2010) Grassland root communities: species distri-

butions and how they are linked to aboveground abundance.

Ecology 91:3201–3209

Hansen B (1972) The genus Balanophora J. R. & G. Forster a

taxonomic monograph. Dansk Botanisk Arkiv 28:1–188

Hansen B (1982) The Balanophoraceae of the Pacific. Acta Phyto-

taxon Geobot 18:92–102

Hansen B (1999) Balanophora species published 1971–1998, mostly

from China and Japan. Nord J Bot 19:641–642

Hatushima S (1971) A new noteworthy species of Balanophora from

Kyushu. J Geobot 19:60–62

Horikawa Y (1972) Atlas of the Japanese flora—an introduction to

plant sociology of East Asia. Gakken Co., Tokyo

Horikawa Y (1976) Atlas of the Japanese flora II—an introduction to

plant sociology of East Asia. Gakken Co., Tokyo

Huang S, Murata J (2003) Balanophoraceae. Flora of China, vol 5.

Missouri Botanical Garden, pp 272–276

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference

of phylogenetic trees. Bioinformatics 17:754–755

Kuwada Y (1928) An occurrence of restitution nuclei in the formation

of embryo sacs in Balanophora japonica. Bot Mag Tokyo

42:117–129

Linder CR, Moore LA, Jackson RB (2000) A universal molecular

method for identifying underground plant parts to species. Mol

Ecol 9:1549–1559

Maddison DR, Maddison WP (2000) MacClade 4: analysis of

phylogeny and character evolution. Sinauer Associates,

Sunderland

Minamitani T (2009) Kyushu plant notes (9): the appearance of

Balanophora yakushimensis on the mainland of Kyushu.

J Miyazaki Plant Res 11:7–10

Murata J (1988) Morphology and distribution of Balanophorafungosa J. R. et G. Forst. (Balanophoraceae). J Jpn Bot

63:201–210

Murata J (1990) Agamic species of Balanophora in Japan. Mem Natl

Sci Mus Tokyo 23:43–50

Murata J (1992) Female flowers of Balanophora kiusiana. J Jpn Bot

67:166–168

Nickrent DL, Duff RJ (1996) Molecular studies of parasitic plants

using ribosomal RNA. In: Moreno MT, Cubero JI, Berner D,

Joel D, Musselman LJ, Parker C (eds) Advances in parasitic

research. Junta de Andalucia, Direccion General de Investiga-

cion Agraria, Cordoba, pp 28–52

Nickrent DL, Garcia MA (2009) On the brink of holoparasitism:

plastome evolution in dwarf mistletoes (Arceuthobium, Visca-

ceae). J Mol Evol 68:603–615

Nickrent DL, Starr EM (1994) High-rates of nucleotide substitution in

nuclear small subunit (18S) rDNA from holoparasitic flowering

plants. J Mol Evol 39:62–70

Nickrent DL, Ouyang Y, Duff RJ, de Pamphilis CW (1997) Do

nonasterid holoparasitic flowering plants have plastid genomes?

Plant Mol Biol 34:717–729

Nickrent DL, Blarer A, Qiu YL, Yidal-Russell R, Anderson FE

(2004) Phylogenetic inference in Rafflesiales: the influence of

rate heterogeneity and horizontal gene transfer. BMC Evol Biol

4:40

Nickrent DL, Der JP, Anderson FE (2005) Discovery of the

photosynthetic relatives of the ‘‘Maltese mushroom’’ Cynomo-rium. BMC Evol Biol 5:28

Posada D, Buckley TR (2004) Model selection and model averaging

in phylogenetics: advantages of Akaike information criterion and

Bayesian approaches over likelihood ratio tests. Syst Biol

53:793–808

Sunaryo (1992) Morphologisch-anatomische Untersuchungen an den

Knollen von Balanophora fungosa J. R. & G. Forst. ssp. indica(Arn.) B. Hansen var. globosa (Jungh.) B. Hansen und Balan-ophora elongata Bl. Fachbereichs Biologie. Ph.D. Philipps-

Universitat Marburg, Marburg, p 122

Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony

(*and other methods). Sinauer Associates, Sunderland

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG

(1997) The CLUSTAL-X windows interface: flexible strategies

for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Res 25:4876–4882

Watanabe K (1942) Morphologisch-biologische studien uber Balan-

ophoraceen in Nippon ausgenommen Taiwan (I). J Jpn Bot

18:250–260

Watanabe K, Akuzawa E (1982) Balanophora. In: Satake Y (ed) Wild

flowers of Japan, herbaceous plants, vol 2. Heibonsha, Tokyo,

pp 12–13

Yahara T, Ohba H, Murata J, Iwatsuki K (1986) Taxonomic review of

vascular plants endemic to Yakushima Island, Japan. J Fac Sci

Univ Tokyo III 14:82–83

326 J Plant Res (2012) 125:317–326

123