Bull. Natl. Mus. Nat. Sci., Ser. B, 40(1), pp. 47–53, February 20, 2014

Molecular Phylogenetic Distinction between Taiwan Endemic
Rhynchotechum brevipedunculatum and R. discolor (Gesneriaceae)
Widespread in Subtropical and Tropical Asia
Goro Kokubugata1,*, Koh Nakamura2, Yukiko Saito3, Mina L. Labuguen4,
Aurea Lopez-Feliciano5 and Masatsugu Yokota6
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
3
Department of Education, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903–0213, Japan
4
Community Environment and Natural Resources Ofﬁce, Cauayan, Isabela, Philippines
5
Isabela State University, Echague, Isabela, Philippines
6
Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus,
Senbaru 1, Nishihara, Okinawa 903–0213, Japan*E-mail: gkokubu@kahaku.go.jp(Received 12 November 2013; accepted 18 December 2013)

Abstract Molecular analyses using ITS of nrDNA and trnSG intron of cpDNA were conducted
to test the phylogenetic distinction between Taiwanese endemic Rhynchotechum brevipedunculatum and R. discolor widespread in subtropical and tropical Asia. The analyses revealed the reciprocal monophyly of the two species. This result indicates that R. brevipedunculatum is an independent species from R. discolor, adding molecular support for the taxonomic concept based onmorphology.
Key words : endemic, Gesneriaceae, ITS, Rhynchotechum, Ryukyus, Taiwan, trnSG.

Introduction
The genus Rhynchotechum Blume (Gesneriaceae) consists of 16 species and primarily occurs
in tropical and subtropical Asia (Wang and Wang,
2000). In Taiwan, two Rhynchotechum species
has long been recognized: R. discolor (Maxim.)
B.L.Burtt widely distributing from Japan to New
Guinea (Burtt, 1962) (Fig. 1A) and R. formosanum
Hatusima occurring in China and Taiwan (Wanget al., 1998). In 2000, a new species R. brevipedunculatum J.C.Wang was described as a Taiwanese endemic species (Wang and Wang, 2000)(Fig. 1B).
Rhynchotechum brevipedunculatum and R.
discolor are apparently distinguishable from R.

formosanum by their morphological characters.
On the other hand, R. brevipedunculatum is morphologically similar to R. discolor, but the two
species are distinguished based on some ﬂoral
traits. The former has peduncles less than 2 mm
long, compact simple cyme inﬂorescences, andcalices of 10 to 12 mm long×1.5 to 2.0 mm
wide; while the latter has peduncles more than
18 mm long, loose compound cyme inﬂorescences, and calices of 5 to 8 mm long×0.5 mm
wide (Wang and Wang, 2000). Distribution
ranges of the two species mostly overlapped in
Taiwan, from New Taipei City to Pingtung. Habitatsof the two Rhynchotechum species are moist andslightly shaded environments under broadleaf
forest canopy, and the two species rarely sympat-

Goro Kokubugata et al.

48

Fig. 1. Habits of Rhynchotechum brevipedunculatum and R. discolor. A. R. discolor (GK212). B. R. brevipedunculatum (GK4155). Bars indicate 2 cm.

rically occur (Wang and Wang, 2000). In thisstudy, we conducted molecular analyses to test
the phylogenetic distinction between R. brevipedunculatum and R. discolor based on the internaltranscribed spacer (ITS) region of nuclear ribosomal DNA and a spacer region between 3ʼtrnS
and trnG (trnSG) of chloroplast DNA.
Materials and Methods
Plant materials
We morphologically identiﬁed Rhynchotechum
brevipedunculatum and R. discolor following the
key of Wang and Wang (2000). Fourteen samplesof R. brevipedunculatum from Taiwan Island,and 39 samples (including 15 Japanese, 20 Taiwanese and 4 Philippine samples) of R. discolor
were collected for the molecular analyses (Table 1
and Fig. 2). In the present study, we found two
localities where the two Rhynchotechum species
occurred within 50 m from each other in Shihting(T5) and Sanhsia (T6) of Taiwan (Table 1 andFig. 2). Rhynchotechum formosanum distributedin China and Taiwan (Wang and Wang, 2000)
was selected as an outgroup taxon (Table 1).
Total number of operational taxonomic units was
54 including the 53 ingroup members and an outgroup member (Table 1). Voucher specimens forthe molecular analyses were deposited in the her-barium of the National Museum of Nature andScience (TNS).
DNA extraction, PCR, and sequencing
Genomic DNA was extracted from silica gel driedleaves using the DNeasy Plant Mini Kit (Qiagen,Qiagen, Valencia, CA) following the manufacturerʼs
protocol. The genomic DNA samples were deposited in the Molecular Biodiversity Research Centerof the National Museum of Nature and Science.
The ITS and trnSG regions were ampliﬁed by thepolymerase chain reaction (PCR) using an iCycler(BIO RAD, California, USA). Forward primer
ʻAB101ʼ (5′-ACG AAT TCA AGG TCC GGT GAAGTG TTC G-3′) and reverse primer ʻAB102ʼ (5′TAG AAT TCC CCG GTT CGC TCG CCG TTA
C-3′) were used for ITS (Douzery et al., 1999); andforward primer ʻtrnS (GCU)ʼ (5′-GCC GCT TTAGTC CAC TCA GC-3′) and reverse primer ʻtrnG(UCC)ʼ (5′-GAA CGA ATC ACA CTT TTA CCA
C-3′) were used for trnSG (Hamilton, 1999). Ampliﬁcations were performed using Takara SapphireAmp Fast Master Mix (Takara, Otsu, Japan) with
Sapphire Amp (Takara, Tokyo, Japan). The PCR
proﬁle was 35 cycles of 5 sec at 98°C, 5 sec at
51°C, and 5 sec at 72°C after an initial denaturing
for 3 min at 94°C for both of ITS and trnSG. The
PCR products were checked by electrophoresis
before puriﬁcation using illustra ExoProStar (GEDistinction between Rhynchotechum brevipedunculatum and R. discolor
Table 1.
SpeciesR. brevipedunculatum

R. discolor

R. formosanum

49

Plant materials of Rhynchotechum species investigated in the present study

Collection locality (altitude)
Taiwan: Keelung (45 m)
Taiwan: Taipei, Shihting (122 m)
Taiwan: Taipei, Shihting (100 m)
Taiwan: Taipei, Shihting (100 m)
Taiwan: Taipei, Shihting (100 m)
Taiwan: Taipei, Sanhsia (535 m)
Taiwan: Taipei, Sanhsia (535 m)
Taiwan: Taipei, Sanhsia (494 m)
Taiwan: Miaoli, Shitan (487 m)
Taiwan: Pingtung, Shihtzu (388 m)
Taiwan: Pingtung, Shihtzu (388 m)
Taiwan: Pingtung, Shihtzu (388 m)
Taiwan: Pingtung, Shihtzu (388 m)
Taiwan: Pingtung, Shihtzu (388 m)
Japan: Ryukyus, Nakanoshima Island (100 m)
Japan: Ryukyus, Nakanoshima Island (150 m)
Japan: Ryukyus, Tokunoshima Island, Amagi (140 m)
Japan: Ryukyus, Tokunoshima Island, Isen (140 m)
Japan: Ryukyus, Tokunoshima Island, Isen (132 m)
Japan: Ryukyus, Tokunoshima Island, Isen (180 m)
Japan: Ryukyus, Okinawa Island, Kunigami (400 m)
Japan: Ryukyus, Okinawa Island, Kunigami (150 m)
Japan: Ryukyus, Okinawa Island, Higashi (50 m)
Japan: Ryukyus, Okinawa Island, Higashi (150 m)
Japan: Ryukyus, Okinawa Island, Motobu (300 m)
Japan: Ryukyus, Ishigaki Island (305 m)
Japan: Ryukyus, Ishigaki Island (300 m)
Japan: Ryukyus, Iriomote Island (70 m)
Japan: Ryukyus, Iriomote Island (230 m)
Taiwan: Taipei, Sanchih (837 m)
Taiwan: Taipei, Sanzhi (820 m)
Taiwan: Taipei, Sanzhi (820 m)
Taiwan: Taipei, Sanzhi (820 m)
Taiwan: Keelung (45 m)
Taiwan: Taipei, Shitan (275 m)
Taiwan: Taipei, Shihting (100 m)
Taiwan: Taipei, Sanhsia (427 m)
Taiwan: Taipei, Pingxi (183 m)
Taiwan: Taipei, Pingxi (183 m)
Taiwan: Taipei, Taipei, Shiting (250 m)
Taiwan: Taipei, Shihting (504 m)
Taiwan: Taipei, Shihting (414 m)
Taiwan: Taipei, Wulai (231 m)
Taiwan: Hsinchu, Chienshih (1339 m)
Taiwan: Miaoli, Chienshih (1110 m)
Taiwan: Hsinchu, Wufen (940 m)
Taiwan: Ilan, Nannao (262 m)
Taiwan: Miaoli, Sanyi (371 m)
Taiwan: Pingtung, Wutai (1075 m)
Philippines: Banaue (1712 m)
Philippines: Isabela, Dinapigue (130 m)
Philippines: Aurora, Dipaculao (363 m)
Philippines: Lucena (unknown)
Taiwan: Taipei, Pinglin (350 m)

Abbreviation*

Voucher
no.

T5
T6
T6
T6
T6
T12
T12
T12
T16
T20
T20
T20
T20
T20
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
T1
T2
T2
T2
T3
T4
T5
T6
T7
T7
T9
T10
T10
T11
T13
T14
T15
T17
T18
T19
P1
P2
P3
P4
T8

GK4173
GK4155
GK14982
GK14984
GK14985
GK14226
GK14227
GK14233
GK5177
GK15039
GK15040
GK15047
GK15048
GK15059
GK11337
GK11341
GK12120
GK12478
GK12594
GK12493
GK342
GK8983
GK10073
GK9026
GK196
GK11814
GK212
GK67
GK207
GK8493
GK14938
GK14939
GK14940
GK4172
GK4150GK14990
GK14235
GK14974
GK14973
GK3099GK15119
GK15112
GK10045
GK5215
GK5185GK5120GK6231
GK5153
GK8902GK9655
GK13141
GK13132
GK911
CIP18195

ITS

trnSG

AccessionTypeno. **

AccessionTypeno. **

AB871639
AB871629
AB871633
AB871634
AB871635
AB871636
AB871637
AB871638
AB871640
AB871641
AB871642
AB871630
AB871631
AB871632
AB871590
AB871591
AB871592
AB871593
AB871594
AB871595
AB871596
AB871597
AB871598
AB871599
AB871600
AB871601
AB871602
AB871603
AB871604
AB871620
AB871609
AB871610
AB871611
AB871619
AB871607
AB871616
AB871617
AB871614
AB871615
AB871605
AB871613
AB871612
AB871618
AB871621
AB871623
AB871606
AB871622
AB871608
AB871624
AB871625
AB871628
AB871626
AB871627
AB871643

AB871721
AB871711
AB871715
AB871716
AB871717
AB871718
AB871719
AB871720
AB871722
AB871723
AB871724
AB871712
AB871713
AB871714
AB871672
AB871673
AB871674
AB871675
AB871676
AB871677
AB871678
AB871679
AB871680
AB871681
AB871682
AB871683
AB871684
AB871685
AB871686
AB871702
AB871691
AB871692
AB871693
AB871701
AB871689
AB871698
AB871699
AB871696
AB871697
AB871687
AB871695
AB871694
AB871700
AB871703
AB871705
AB871688
AB871704
AB871690
AB871706
AB871707
AB871710
AB871708
AB871709
AB871725

A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
D
B
B
F

a
b
a
a
a
a
a
a
a
c
c
b
b
b
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
e
d
d
d
e
d
e
e
d
e
d
d
d
e
e
e
d
e
d
e
f
f
f
f
g

* Referring Fig. 2.
** Deposited Sequences in the DDBJ database.

Helthcare, Tokyo, Japan). Cycle sequencing
reaction was carried out with the BigDye Terminator Cycle Sequencing Kit ver. 3.1 (Applied
Biosystems, Foster City, CA) using the same
PCR primers. 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 analysed using ATGC ver. 4.01

50

Goro Kokubugata et al.

Fig. 2. Map showing the 39 collection localities of the 54 individuals of three Rhynchotechum species from the
Ryukyus of Japan, Taiwan, and the Philippines. Abbreviations refer to localities listed in Table 1.

(GENETYX Co., Tokyo, Japan). Sequence datafrom this study were deposited to the DDBJ database (http://www.ddbj.nig.ac.jp/) (Table 1).
Phylogenetic analysis
DNA sequences were aligned using the program ClustalW 1.8 (Thompson et al., 1994) andthen manually adjusted. The combinability
between the ITS and trnSG regions was assessedusing the incongruence length difference (ILD)
test (Farris et al., 1994).
Phylogenetic analyses were conducted basedon a Bayesian approach using MrBayes 3.1.2(Ronquist and Huelsenbeck, 2003) and a maximum parsimony (MP) criterion using PAUP*
version 4.0b10 (Swofford, 2002). In the Bayesian
phylogenetic analysis, the Hierarchical Likelihood Ratio Tests (hLRT) implemented in
MrModeltest 2.2 (Nylander, 2004) was used toestimate the appropriate evolutionary model of

nucleotide substitutions. Based on the model
selected, two separate runs of Metropolis coupled Markov chain Monte Carlo (MCMCMC)
analyses were performed, each with a random
starting tree and four chains (one cold and threeheated). The MCMCMC length was two million
generations, and the chain was sampled every
one hundredth generation from the cold chain.
The ﬁrst 5000 sample trees (25% of the total
20000 sample trees) were discarded as burn-in
after checking that the average standard deviation of split frequencies (ASDSF) reached a stationary state at ＜0.01 thereafter. As a guide toconvergence, the potential scale reduction factors(PSRFs) were ascertained to be reasonably close
to 1.0 for all parameters in an output table. A
50% majority consensus tree of the output treeﬁle from MrBayes was generated by TREEVIEW (Page, 1996).
In the MP phylogenetic analysis, characters

Distinction between Rhynchotechum brevipedunculatum and R. discolor

were treated as unordered, and character transformations were equally weighted. Indels werescored as binary states indicating presence orabsence, following the simple indel-coding strategy of Simmons and Ochoterena (2000). The
branch collapse option was set to collapse at aminimum length of zero. A heuristic parsimony
search was performed with 200 replicates of random additions of sequences with ACCTRAN
character optimization, tree bisection–reconnection (TBR) branch swapping, and MULTREES
and STEEPEST DESCENT options on. Statistical support for each clade was assessed by bootstrap analysis (Felsenstein, 1985). A thousand
replicates of heuristic searches, with the TBR
branch swapping switched on and MULTREES
options off, were performed to calculate bootstrap values (BS).
Results and Discussion
Phylogenetic distinction based on ITS and trnSG
After alignment of the sequences from the 54
samples, a matrix of 636 bp was obtained for
ITS. Four ITS types (A–D) were recognized in
the ingroup; only type A was found in R. brevipedunculatum and the other three types B–D were
found in R. discolor (Table 1). A matrix of 704 bp
was obtained for trnSG after alignment. Six haplotypes (a–f) were recognized in the ingroup;
three haplotypes (a–c) were found in R. brevipedunculatum and the other three haplotypes (d–f)
were found in R. discolor (Table 1). The ILD test
did not show signiﬁcant incongruence between
the two regions (p＝1.00), and thus they werecombined for the present Bayesian and MP analyses. The length of the combined matrix was
1340 bp.
The model of F81 was selected in the Bayesian analysis. The 50% majority rule consensus
tree of all the post-burn-in trees is depicted withmean branch lengths and posterior probabilities(PP) (Fig. 2). In the MP analysis, 14 of 26 variable characters were parsimony-informative, andtwo equally parsimonious trees of 26 steps wereobtained with CI of 1.000 and RI of 1.000.

51

Because the topology of the MP strict consensus
tree (not shown) was compatible with that of theBayesian 50% majority rule consensus tree, thebootstrap percentages (BP) were plotted on theBayesian tree (Fig. 3).
The Bayesian and MP analyses (Fig. 3) showed
two clades in the ingroup: one composed of all the
14 plants of R. brevipedunculatum (clade I; PP/
BS＝1.00/86%) and the other clade composed of
all the 39 plants of R. discolor (clade II; PP/BS＝1.00/87%). In clade I, twelve plants of R. brevipedunculatum formed a subclade (PP/BS＝0.55/—%). In clade II, 10 Taiwanese plants of R. discolor formed a subclade (PP/BS＝1.00/85%).
As mentioned in the introduction, R. brevipedunculatum is morphologically similar to R. discolor (Wang and Wang, 2000). In addition, during sample collection, we found that the two
species occurred almost sympatrically within
50 m from each other in the two localities (T5and T6) in Taiwan. However, the present molecular analyses revealed that the two species wereclearly separated. This result indicates thatR. brevipedunculatum is an independent species
from R. discolor, adding molecular support for
Wang and Wang (2000).
In the present analyses, no hybrid of the two
species was found, although it was expectedfrom their sympatric occurrence. Therefore, there
likely is some barrier to the genetic exchange
between the two species. The corolla tube of
R. brevipedunculatum (ca. 2 mm) is longer thanthat of R. discolor (ca. 1 mm) (Wang and Wang,
2000). It is possible that the two species have different pollinators, because of which they are
reproductively isolated.
Molecular polymorphism of R. brevipedunculatum and R. discolor
In the present molecular survey, both R. brevipedunculatum and R. discolor had multiple ITS/
trnSG types. However, the geographical distribution of the different ITS/trnSG types did not correlate with geographical coordinate or elevationof the sampling localities (Table 1). Additionally,in two localities T6 and T20 in Taiwan, R. brevi-

Goro Kokubugata et al.

52

Fig. 3. The Bayesian 50% majority rule consensus tree of Rhynchotechum based on nrITS and cpDNA trnSG
sequences. The topology of the maximum parsimony strict consensus tree was compatible with the Bayesiantree. Alphabets in parentheses indicate nrITS and trnSG types. Numerals above branches indicate Bayesianposterior probabilities (left) and bootstrap percentages in the maximum parsimony analysis (right; –<50%).

pedunculatum had intrapopulation polymorphismin trnSG. Rhynchotechum species have fresh fruits(Wang and Wang, 2000), which are expected tobe dispersed by birds. The absence of geographicalpattern in the ITS/trnSG type distribution and theintrapopulation polymorphism may be caused by

Distinction between Rhynchotechum brevipedunculatum and R. discolor

high mobility of fruits/seeds via bird dispersal.
To further discuss the phylogeography of the two
species, more detailed analyses with dense sampling of populations are needed.
Acknowledgments
We thank C.-I Peng for providing materials.
This study was supported in part by the JSPS
Grants-in-Aid for Scientiﬁc Research, Program C(No. 20510220) and the Mitsui & Co., Ltd. Environment Fund (No. RC10–C097).
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