Bull. Natl. Mus. Nat. Sci., Ser. B, 39(2), pp. 95–100, May 22, 2013

Migration of Lobelia zeylanica (Campanulaceae) from Taiwan to theRyukyu Archipelago Inferred from Chloroplast DNA Data
Goro Kokubugata1,*, Yukiko Saito1, Koh Nakamura2, Aurea Lopez-Feliciano3,
Mina L. Labuguen4 and Masatsugu Yokota5
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
3Isabela State University, Echague, Isabela, Philippines
4Community Environment and Natural Resources Ofﬁce, Cauayan, Isabela, Philippines
5Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus,
Nishihara, Okinawa 903–0213, Japan*E-mail: gkokubu@kahaku.go.jp(Received 12 February 2013; accepted 25 March 2013)

Abstract Molecular phylogenetic analysis of Lobelia zeylanica, broadly distributed in tropicalAsia, were conducted to elucidate its migration route to the northernmost populations in subtropic
Yonaguni Island, the Ryukyu Archipelago of Japan. The present analysis using rbcL gene andtrnL-F intergenic spacer region of chloroplast DNA revealed that all two Yonaguni plants and threeof four Taiwanese plants shared the identical sequences in the two markers. They formed a wellsupported clade with a Taiwanese remainder and were separated from plants from Malesian
regions. The present study suggests that L. zeylanica have likely migrated from Taiwan to Yonaguni Island.
Key words : Lobelia, migration, phylogeography, Ryukyus, Taiwan.

Introduction
The Ryukyu Archipelago (the Ryukyus) is situated between Kyushu Island of Japan and Taiwan, and comprises about 140 islands (Fig. 1).
The Ryukyus is biogeographically divided into
three major areas, namely, the northern, central
and southern Ryukyus, each of which has a characteristic biota (e.g., Tagawa and Miyagi, 1991;Ota, 1998; Nakamura et al., 2009). Yonaguni
Island studied herein belongs to the southern
Ryukyus, and is about 130 km away from Taiwan
Island (Fig. 1). Yonaguni and Taiwan islands
share many ﬂoristic elements including those
presumably originated in Malesian region(Hatusima, 1975), but few phylogeographicalstudy has been conducted to elucidate migratory
history of such plant taxa.

Lobelia zeylanica L. (Campanulaceae, sect.
Delostemon) (Fig. 2) is a perennial herb, andwidely distributed from New Guinea and SriLank (Lammers, 2007) to Yonaguni Island of theRyukyus (Hatusima, 1975). In Japan, this species
is known from only three populations in Yonaguni Island (unpublished data), which marks thenorthern limit of its distribution range. Thus L.
zeylanica is treated as a critically endangered
species in the Japanese plant redlist (JapaneseMinistry of the Environment, 2012). In thisstudy, we conducted molecular phylogenetic
analysis on L. zeylanica collected from Yonaguni
Island, Taiwan Island, Luzon Island of the Philippines and Borneo Island of Malaysia, using rbcL
gene and trnL-F intergenic spacer region of chloroplast DNA for discussing its migration route toYonaguni Island.

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Materials and Methods
Plant materials
For the present molecular analysis, a plant ofLobelia zeylanica was collected from each population at two localities in Yonaguni Island, four
localities in Taiwan Island, one locality in Luzon
Island of the Philippines, and two localities inBorneo Island (Malaysian territory), in total nine
plants were obtained (Table 1 and Fig. 1).
Voucher specimens for the molecular analysis
were deposited in the herbarium of the National
Museum of Nature and Science (TNS).
Lobelia aquatica Cham. closely related to L.
zeylanica (Kokubugata et al., 2012), wasselected as an outgroup taxon, and its sequencesof rbcL (EF141029) and trnL-F (DQ356182)
reported by Antonelli (2008) were obtained fromthe DDBJ/EMBL/GenBank database (http://www.ncbi.nlm.nih.gov/gquery/) (Table 1).
Fig. 1. Map showing nine localities where Lobelia
zeylanica plants were collected. Broken lines
circle the islands of the Ryukyus. For abbreviations for collection localities, refer Table 1.

Fig. 2.

DNA extraction, PCR and sequencing
Genomic DNA was extracted from silica gel

Habit of Lobelia zeylanica in Yonaguni Island (GK14127; photographed on October 11, 2011).

Migration of Lobelia zeylanica from Taiwan
Table 1.
SpeciesL. zeylanica

97

Nine plants of Lobelia zeylanica investigated in the present study

Collection locality

Abbreviation*

Voucherno.

YN1

Japan, Ryukyus, Okinawa,Yonaguni (1)Japan, Ryukyus, Okinawa,
Yonaguni (2)Taiwan, New Taipei, Pingchi
Taiwan, New Taipei, Shihting
Taiwan, Ilan, TouchengTaiwan, Nantou, Yuchih
Philippines, Luzon, Isabela,
Dinapigue
Malaysia, Borneo, Sabah, Kinabaru
Malaysia, Borneo, Sabah, Crocker

rbcL

trnL-F
Type

Accession
no.**

Type

AB818960

A

AB793518

aGK14127A

AB818961

A

AB793519

aGK8775
GK15118
GK14959
GK11617

AB645971
AB818963
AB818962
AB818964

AA
AA

AB793522
AB793521
AB793520
AB793523

aaab

LZ

GK13156

AB818965

BAB793524

cBN1
BN2

GK7894
GK7880AB645970
AB645969

CC

AB793525
AB793526

deAccessionno.**GK14126

YN2
TW1TW2TW3TW4* Referring Fig. 1.
** Deposited Sequences in the DDBJ database.

dried leaves using the DNeasy Plant Mini Kit(Qiagen, Qiagen, Valencia, CA) following themanufacturerʼs protocol. The genomic DNA samples were deposited in the Molecular Biodiversity Research Center of the National Museum ofNature and Science, Japan. The sequence of
1,5-bisphosphate carboxylase/oxygenase largesubunit gene (rbcL) and intergenic spacer region
between trnL and trnF genes including
trnL3′exon (trnL-F) were ampliﬁed by the polymerase chain reaction (PCR) using an iCycler(BIO RAD, California, USA). Forward primer
ʻrbcLcFʼ (5′-TGA AAA CGT GAA TTC CCA
ACC GTT TAT GCG-3′) and reverse primer
ʻrbcLaRʼ (5′-CTT CTG CTA CAA ATA AGA
ATC GAT CTC TCC A-3′) were used for rbcL
(Hasebe et al., 1994); and forward primer
ʻtrnT UGUF (TabA)ʼ (5′-CAT TAC AAA TGC GATGCT CT-3′) and reverse primer ʻtrnF GAA (TabF)ʼ(5′-ATT TGA ACT GGT GAC ACG AG-3′) wereused for trnL-F (Taberlet et al., 1991). Ampliﬁcations were performed using Takara Ex Taq(Takara, Otsu, Japan) with Ampdirect Plus (Shimadzu, Kyoto, Japan). The PCR proﬁle was 35
cycles of 30 sec at 94°C, 30 sec at 50°C, and1.5 min at 72°C after an initial denaturing for
3 min at 94°C for rbcL; and 35 cycles of 30 sec at94°C, 30 sec at 55°C, and 1.5 min at 72°C after an
initial denaturing for 3 min at 94°C for trnT-F.
The PCR products were checked by electropho-resis before puriﬁcation using ExoSAP-IT (USBCorp., Ohio, USA). Cycle sequencing was carried out using the BigDye Terminator Cycle
Sequencing Kit ver. 3.1 (Applied Biosystems,Foster City, CA); using the PCR primers
for rbcL, and using PCR primer of ʻtrnF GAA(TabF)ʼ, and additional internal forward primers
ʻtrnL59UAAF (TabC)ʼ (5′-CGA AAT CGG TAG
ACG CTA CG-3′) and 39trnLUAAF (TabE) (5′GGT TCA AGT CCC TCT ATC CC-3′) and areverse primer ʻ39trnLUAAR (TabD) (5′-GGG
GAT AGA GGG ACT TGA AC-3′) for trnL-F(Taberlet et al., 1991). The Sanger sequencing
products were then puriﬁed by ethanol precipitation. Automated sequencing was carried out on
an Applied Biosystems 3130xl Genetic Analyzer(Applied Biosystems, California, USA). The
electropherograms were assembled using ATGC
ver. 4.01 (GENETYX Co., Tokyo, Japan).
Sequence data from this study were deposited toDDBJ (http://www.ddbj.nig.ac.jp/) (Table 1).
Phylogenetic analysis
DNA sequences were aligned using ClustalW
1.8 software (Thompson et al., 1994) and thenmanually adjusted. The combinability of the two
cpDNA regions was assessed using the incongruence length difference (ILD) test (Farris et al.,1994). Phylogenetic analysis were conductedbased on a maximum parsimony (MP) criterion

Goro Kokubugata et al.

98

using PAUP* version 4.0b10 (Swofford, 2002).
In the MP phylogenetic analysis, indels weretreated as missing data. Characters were treatedas unordered, and character transformations wereweighted equally. The branch collapse option
was set to collapse at a minimum length of zero.
A heuristic parsimony search was performedwith 200 replicates of random additions ofsequences with ACCTRAN character optimization, tree bisection–reconnection (TBR) branchswapping, and MULTREES and STEEPESTDESCENT options on. Statistical support foreach clade was assessed by bootstrap analysis(Felsenstein, 1985). Ten thousand replicates ofheuristic searches, with the TBR branch swapping, and MULTREES option off, were performed to calculate bootstrap values (BS).
Results and Discussion
Phylogenetic relationships based on rbcL andtrnL-F
In rbcL sequence, three haplotypes were recognized (types A–C) in Lobelia zeylanica,including type A in two plants from Yonaguni
Island and the four plants from Taiwan Island,type B in the plant from Luzon Island, and type
C in the two plants from Borneo Island (Table 1and Fig. 3). In trnL-F sequence, ﬁve haplotypes
a–e were recognized (types a–e), including type ain the two plants from Yonaguni Island and threeof the four plants from Taiwan Island, type b in
the other plant from Taiwan Island, type c in theplant from Luzon Island, type d in the plant fromBorneo Island, and type e in the other plant fromBorneo Island (Table 1 and Fig. 3).
After alignment of the sequences of the nine
plants of L. zeylanica with the outgroup taxon ofL. aquatica (in total 10 OTUs), the sequencelength of 867 bp in rbcL and 972 bp in trnL-F
were obtained. The ILD test did not show signiﬁcant incongruence between the two markers( p＝1.00). The MP analysis was therefore conducted using the combined data of 1839 (867＋972) bp. In the MP analysis, 4 of 28 (3 of 7 inrbcL and 1 of 21 in trnL-F) variable characters
were parsimony-informative. A single parsimonious tree of 28 steps was obtained with a consistency index (CI) of 1.00 and retention index (RI)of 1.00.
Nine plants of L. zeylanica were divided tothree clades (I to III in Fig. 3): clade I was composed of plants from Yonaguni and Taiwan
islands, clade II was composed of the plant fromLuzon Island, and clade III was composed of thetwo plants from Borneo Island.

Fig. 3. The most parsimonious tree for nine plants of Lobelia zeylanica with an outgroup taxon of L. aquatica.
Numbers above the branches indicate bootstrap percentage. Parenthetic abbreviations and alphabets indicate
collection locality (refer Table 1 and Fig. 1) and haplotypes of rbcL (A–C) and trnL-F (a–e).

Migration of Lobelia zeylanica from Taiwan

Implication of dispersal event of L. zeylanica
from Taiwan to Yonaguni Island
As mentioned in the introduction, L. zeylanica
primarily distributes in the Asian tropics (e.g.,Lammers, 2007), with extending subtropical
Yonaguni Island as northern limit of its distribution range (Hatusima, 1975). Therefore, it ismore reasonable to consider that this species has
dispersed from south to north in the north hemisphere. Our MP analysis revealed that the plantsof L. zeylanica from Yonaguni Island were moreclosely related to those from Taiwan Island thanthose from Luzon and Borneo islands. Based onthese results, we conclude that L. zeylanica has
likely migrated from Malesian region to Yonaguni Island via Taiwan Island.
Lobelia zeylanica is known to bear dehiscentdry fruits and seeds with no attachment like awing (Lammers, 1998). Therefore, this species isnot likely to cross over sea migratory birds (fruit)or winds (seed). Geohistorically, Yonaguni Islandis thought to have been ﬁnally separated fromTaiwan Island in early Pleistocene (1.5–0.7MYA) based on geological and palaeo-oceanological data (Koba, 1992; Osozawa et al., 2011).
Substitution rate of trnL-F spacer region in
annual or perennial herbs was estimated as 8.24
×10–9 (substitution/site/year) (Richardson et al.,2001). The timing of the ﬁnal disconnection
between Yonaguni and Taiwan islands and theidentical sequences of trnL-F of L. zeylanica
between Yonaguni Island and Taiwan Island arecompatible with vicariance scenario. Therefore,
we can hypothesize that L. zeylanica may havedispersed through land bridge before early Pleistocene in a case of migration from Taiwan Island
to Yonaguni Island.
However, L. zeylanica widely distribute intropical Asia (Lammers, 2007), and it is difﬁcult
to explain all dispersal events through landbridge for this species. It is reported that certain
Lobelia species have both of dry and berry fruits,
for example fruits of L. angulata G. Forst. werevaried from berry to capsule (Moelino and Tuyn,1960); and berry and dry capsule fruits of L. ﬂuviatilis R.Br. (＝Hypsela sessiliﬂora F.E. Wim-99mer) were respectively reported by Wimmer(1953) and Heenan et al. (2008). Therefore, it ispossible to assume that L. zeylanica may rarely
have had berry fruit, and have been dispersed
oversea by migrate birds as the step-wise colonization outlined by Chiang and Schaal (2006). If
the dispersal event has happened in this species,
we can also assume oversea migration from Taiwan Island to Yonaguni Island after separation ofYonaguni Island from Taiwan Island. Further
fruit morphological surveys must be necessaryfor discussing this issue.
Acknowledgments
We thank C.-I Huang, M. Matsumura, C.-I
Peng and M. Suleiman for assistances in the ﬁeldtrips. This study was conducted in connection
with the projects ʻElucidative studies of delimitation and origin on endemic and narrow-range
species in Japanʼ and ʻIntegrated research on biodiversity of interspecies relationshipsʼ managed
by the National Museum of Nature and Science;
was partly supported by the Mitsui & Co., Ltd.
Environment Fund (No. C10-C097) and JSPS
KAKENHI (Grant No. 25290085).
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