Bull. Natl. Mus. Nat. Sci., Ser. B, 37(1), pp. 9–22, February 22, 2011

Taxonomic Reconsideration and Phylogeographic Implication
for Nertera yamashitae (Rubiaceae)
Goro Kokubugata1, *, Koh Nakamura2, Yumiko Hirayama1,
Ching-I Peng2 and Masatsugu Yokota3
1

Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305–0005 Japan
2
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
3
Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus,
Nishihara, Okinawa, 903–0213 Japan* E-mail: gkokubu@kahaku.go.jp(Received 16 November 2010; accepted 22 December 2010)

Abstract Anatomical and morphological characters and phylogenetic position of Nertera yamashitae were investigated with those of related species in the family Rubiaceae. In the anatomy
and morphology, disk morphology and number of ovules in an ovary locule of N. yamashitae
differed from those of other Nertera species, and were more similar to those of Ophiorrhiza
species. In the sequence similarity search of ITS in nrDNA, and atpB–rbcL and trnK/matK in
cpDNA, N. yamashitae showed the highest similarity to Ophiorrhiza species. In the MP phylogenetic analyses based on nrITS, atpB–rbcL, and trnK/matK, N. yamashitae was outside of a clade of
the genus Nertera in tribe Anthospermeae, while it was included in a clade of the genus Ophiorrhiza in tribe Ophiorrhizeae. The present study, in taxonomy, suggests that N. yamashitae shouldbe treated as a species in Ophiorrhiza as a new combination; and in phylogeography, implies that it
could be a relict species lineage in Amami Island of the Ryukyu Archipelago.
Key words : dwarfism, Nertera, Ophiorrhiza, Rubiaceae, Ryukyus.

Introduction
The genus Nertera Banks et Sol. ex Gaertn. iscomposed of ca. 16 (or six in a strict sense)
species found in Malesia, Oceania and Americas;
and is classified in tribe Anthospermeae (subfamily Rubioideae), being well supported bymorphological and molecular data (Bremekamp,
1966; Bremer and Manen, 2000; Bremer, 2009).
Although Heads (1996) included Nertera species
in the genus Coprosma, disagreeing with mosttaxonomic treatments, he still recognized it as ataxon at section level, namely sect. Nertera. As
the first species of the genus Nertera discoveredin Japan, Nertera yamashitae T. Yamazaki (Fig.
1) studied herein was described based on a typespecimen collected from Amami Island in the
Ryukyu Archipelago (Yamazaki, 1998), because

it has small leaves, four stamens, and a fourlobed corolla, showing the generic-representative
morphologies for Nertera (e.g. Lo, 1999; Liu andYang, 1998). This species has been thought to be
an insular endemic species of Amami Island, andonly three populations have been found in tworiver systems in this island (Hotta, 2004). Therefore, it is treated as critically endangered by theJapanese Ministry of the Environment (2007).
After Yamazaki’s description (1998), although a
few studies on cytology (Kokubugata et al.,1998) and conservation biology (Hotta, 2004)
have been carried out, further taxonomical reconsideration based on anatomical, morphological,
and molecular data has never been applied to thisspecies.
Dwarfism, a phenomenon in which body size
is strongly reduced, is well-known in alpine

10

Goro Kokubugata et al.

Fig. 1. Nertera yamashitae. (A) Habit (photographed by H. Yamashita); (B) flower in plants (GK11029) cultivated in Tsukuba Botanical Garden, National Museum of Nature and Science. Bar5 mm.

ecosystems (e.g. Körner, 1999). In the Ryukyus,
some leaf dwarfisms are known at elevations over
1,000 m on Yakushima Island in the northern
Ryukyus, and phylogenetic and their evolutionary biology has been examined in these plants(e.g. Yokoyama et al., 2003; Shinohara andMurakami, 2006; Tsukaya et al., 2007). Also,
certain dwarf leaf species are known at altitudesof less than 100 m on Amami Island, such as N.
yamashitae studied herein, Lysimachia liukiuensis Hatus. (Myrsinaceae), Oxalis exilis A. Cunn.
(Oxalidaceae), Rubus amamianus Hatus. et Ohwi
(Rosaceae), and Sphenomeris minutula Sa. Kurata (Lindsaeaceae) (Hatusima, 1975). In contrast
with leaf dwarfism at high elevation on Yakushima Island, however, few phylogenetic studies
have examined for the dwarf leaf species at low
altitude on Amami Island without Lysimachia
liukiuensis (Kokubugata et al., 2010).
In the present study, we investigated the floral
anatomy and morphology of N. yamashitae; andits phylogenetic position among related speciesof subfamily Rubioideae based on three molecular loci to reconsider the taxonomic status, and to
discuss the dwarfism of this species.

Materials and Methods
Plant materials
Plants of Nertera yamashitae were collectedfrom two localities in two river systems in
Amami Island (Table 1). Also, plants of N.
granadense (Mutis ex L.f.) Druce following astrict taxonomic treatment of Liu and Yang
(1998) in four Asian countries, and two Ophiorrhiza species in two Asian countries were collected and used in the present molecular phylogenetic analyses (Table 1). Voucher specimens were
deposited in the herbarium of the National Museum of Nature and Science (TNS).
DNA extraction, polymerase chain reaction, andsequencing
Fresh leaves were harvested from the plants
cultivated in a greenhouse of the Tsukuba Botanical Garden for DNA extraction, which was conducted using the DNeasy Plant Mini Kit (Qiagen,
Valencia, CA) following the manufacturer’s protocols. Total DNA samples isolated were deposited in the Molecular Biodiversity Research Centerof the National Museum of Nature and Science.

GK: G. Kokubugata (TNS). b Sequences investigated here and registered in the DDBJ/EMBL/GenBank database; letters in parentheses indicate the locus type.

Ophiorrhiza longiflora Blume
Ophiorrhiza sp.

N. granadense (Mutis ex L.f.) DruceaAB559550(I)AB559551(I)
—
—
—
—
—
—
—
—
AB559552
AB559553
AB559538 (a)AB559539 (a)AB559540 (b)AB559541 (c)AB559542 (d)AB559543 (e)AB559544 (f)AB559545 (g)AB559546 (h)AB559547 (i)AB559548
AB559549
AB559526 (A)AB559527 (A)AB559529 (B)AB559528 (B)AB559530 (C)AB559531 (D)AB559532 (E)AB559533 (E)AB559534 (F)AB559535 (G)AB559536
AB559537
GK9854
GK11029
GK11028
GK8824
GK10821
GK8301
GK8254
GK9647
GK7903
GK8559GK8503
GK12391
JAPAN, Ryukyus: Naze, Amami Is., Kagoshima
JAPAN, Ryukyus: Sumiyo, Amami Is., Kagoshima
TAIWAN: Nanao, Ilan
TAIWAN: Taoyuan, Kaohsiung
TAIWAN: Taoyuan, Kaohsiung
PHILIPPINES: Bontoc, Mountain Province, Luzon
PHILIPPINES: Trinidad, Benguet, Luzon Is.
PHILIPPINES: Banaue, Ifuga, Luzon Is.
MALAYSIA, Borneo: Kinabaru, Sabah
INDONESIA, West Java: Cibodas, Cianjur
INDONESIA, West Java: Cibodas, Cianjur
THAILAND: Ang Kang, Chiang Mai

Voucher
Locality
Species

Nertera yamashitae T. Yamaz.

trnK/matK (type)atpB–rbcL (type)
ITS (type)

Accession no.ba

Table 1. Plant materials of Nertera and Ophiorrhiza investigated in the present study, and their localities, voucher numbers, and ITS, atpB–rbcL and trnK/matKGenBank accession numbers

Taxonomy and Phylogeography of Nertera yamashitae

11

In the present study, ITS1, 5.8S rDNA andITS2 with part of the 18S rDNA (ITS) of nuclear
ribosomal DNA (nrDNA), the intergenic spacerof atpB and rbcL genes (atpB–rbcL), thematuraseK gene and adjacent trnK introns(trnK/matK) of cpDNA were selected for themolecular phylogenetic analyses. In the polymerase chain reaction (PCR), we amplified
ITS using the primer set AB101 and AB102(Douzery et al., 1999), atpB–rbcL using atpB2F
and rbcL2R (Nakamura et al., 2006), trnK/matKusing trnK–3914(D)F (Johnson and Soltis, 1994)
and psbA5R (M. Nepokroeff, University ofSouth Dakota, personal communication). The
PCR amplifications were performed using Takara
Ex Taq (Takara, Tokyo) with Ampdirect Plus
(Shimadzu, Kyoto) on an iCycler (BioRad, Hercules, CA). The PCR profile comprised 35 cyclesof 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C
for ITS, and 35 cycles of 30 s at 94°C, 30 s at
55°C, and 3 min at 72°C for atpB–rbcL andtrnK/matK. After purification of PCR products
using ExoSAP–IT (USB Corp., Cleveland, OH),
direct sequencing was performed using the
BigDye Terminator Cycle Sequencing Kit ver. 3(ABI PRISM; Applied Biosystems, Foster City,CA), with the PCR primers AB101 and AB102,
and two additional primers ITS2 and ITS3
(White et al., 1990) for the ITS; the PCR primers
atpB2F and rbcL2R for atpB–rbcL; and the PCRprimers trnK–3914 (D) and psbA5R and additional primers of matK–300F (Yamashita andTamura, 2000), trnK2621R (Liston and Kadereit,1995), matK–AF, matK–8R (Ooi et al., 1995),
and matK–AR (Kazempour Osaloo et al., 1999)
for matK/trnK. Automated sequencing was performed on a 3130xl Genetic Analyzer (Applied
Biosystems). The sets of sequence data wereassembled using ATGC ver. 4.01 (GENETYX
Co., Tokyo). Sequence data of ITS, atpB–rbcL,
and trnK/matK were deposited in the DDBJ/
EMBL/GenBank international DNA database
under the accession numbers shown in Table 1.

12

Goro Kokubugata et al.

Sequence similarity searches and taxon sampling
in DNA database
Sequence similarity searches in the ITS,
atpB–rbcL, and matK/trnK regions of N. yamashitae were conducted with the program Basic
Local Alignment Search Tool (BLAST) ver.
2.2.18 (Altschul et al., 1997) in the DDBJ/
EMBL/GenBank database (http://blast.ddbj.nig.
ac.jp/top-j.html) on 23 February 2010. Following
the BLAST results, we selected tribe membersthat showed high similarity to N. yamashitae inthe examined three regions, and their sequence
data deposited in the DNA database were added
to the present phylogenetic analyses under theaccession numbers shown in Table 2.
Phylogenetic analysis
DNA sequences were aligned using the program ClustalX 1.8 (Thompson et al., 1994) andthen manually adjusted. Phylogenetic analyses
were conducted based on a maximum parsimony(MP) criterion using PAUP* version 4.0b10(Swofford, 2002). First, to verify the phylogenetic
position of N. yamashitae at the tribe level, MP
phylogenetic analyses based on ITS and
atpB–rbcL were independently conducted withmembers of tribes Anthospermeae including
Nertera species, Ophiorrhizeae including Ophiorrhiza, and Urophylleae, which is thought to bea sister group of tribe Ophiorrhizeae (Rydin etal., 2009a, 2009b). Second, to elucidate the phylogenetic position of N. yamashitae among phylogenetically closely related species, MP phylogenetic analysis was conducted based on combined data of ITS, atpB–rbcL, and trnK/matK.
The combinability of the two cpDNA regions
was assessed using the incongruence length difference (ILD) test (Farris et al., 1994), with thepartition homogeneity test implemented in
PAUP* version 4.0b10 (Swofford, 2002). Indels
were treated as missing data. Characters weretreated as unordered, and character transformations were weighted equally. The branch collapse
option was set to collapse at a minimum lengthof zero. A heuristic parsimony search was performed with 200 replicates of random additionsof sequences, with the ACCTRAN character
optimization, tree bisection–reconnection (TBR)
branch swapping, MULTREES, and STEEPESTDESCENT options on. Statistical support foreach clade was assessed by bootstrap analysis(Felsenstein, 1985). One thousand replicates ofheuristic searches, with the TBR branch swapping and MULTREES options off, were performed to calculate bootstrap values. Strict consensus trees of the output tree files from PAUP
were generated by TREEVIEW (Page, 1996).
Floral anatomical and morphological observations in Nertera yamashitae
Corolla morphology of N. yamashitae wasobserved based on living plants and herbarium
specimens (GK 9854 and 11029 in TNS). Number of ovules was counted on a mature flower(GK 11029) fixed in FAA solution after cultivation in the Tsukuba Botanical Garden of National
Museum of Nature and Science. The ovary wastorn using a pair of tweezers, the number ofovules counted, and their position observed andphotographed under a light microscope.
Results
Sequence similarity searches of ITS, atpB–rbcL,
and matK/trnKIn two plants of Nertera yamashitae collectedfrom two different river systems on Amami Island, no intraspecific sequence variation was detected in the three sequences of ITS, atpB–rbcL,
and trnK/matK. The results of the sequence similarity searches using BLAST are summarized inTable 3. The BLAST analyses indicated that ITS(684 bp) of N. yamashitae had highest similarity
to 18 accessions of six Ophiorrhiza species inJapan and Taiwan. An accession of N. dichondrifolia Hook. f. (DQ501279; Wright et al., 2006)
was 91st in the BLAST list. The atpB–rbcL (1149bp) of N. yamashitae had the highest similarity to
17 accessions of six Ophiorrhiza species inJapan and Taiwan. An accession for atpB–rbcL
of Nertera sp. (DQ131755; J. F. Manen, unpubl.
data) was 78th in the BLAST list. The trnK/matKOphiorrhiza hayatana Masam.

Ophiorrhiza japonica Bl.

Anthospermum herbaceum L.f.
Nertera granadensis (Mutis ex L.f.) Druce
Normandia neocaledonica Hook.f.
Lerchea bracteata Valeton
Neurocalyx championii Benth. ex Thwaites
Neurocalyx zeylanicus Hook.
Ophiorrhiza elmeri Merr.
Ophiorrhiza mungos L.
Ophiorrhiza amamiana (Hatus.) Koh Nakam.,Denda, Kameshima et Yokota

Anthospermeae

Ophiorrhizeae

Species

TribeaBremer 3039(UPS)—
Munzinger 532(MO)Axelius 343(S)Thor 601(S)B & Bremer 937(S)Kjeldsen & Poulsen 233(AAU)
Bremer 2201(UPS)NK061455(RYU)
NKT0126(RYU)
NK061456(RYU)
NKT0128(RYU)
NKT0129(RYU)
NK061458(RYU)
NKT0127(RYU)
NK061457(RYU)
NKT01015(RYU)
NKT0104(RYU)
NKT0107(RYU)
NKT0111(RYU)
NK061419(RYU)
NK050501(RYU)
NKT0113(RYU)
NK050790(RYU)
NKT0118(RYU)
NKT0120(RYU)
NKT0119(RYU)
NKT0121(RYU)
NKT0122(RYU)
NKT0123(RYU)
NKT0124(RYU)
GK3989(TNS)NKG001(RYU)
NKC001(RYU)
NKC002(RYU)
NKC004(RYU)

Voucher
EU145355
AF2579274
AF2579304
EU1453741
EU1453761
EU1453751
EU1453781
EU1453771
AB2698755(H)AB2698755(H)AB2698755(H)AB2698755(H)AB2698755(H)AB2698755(H)AB2698765(I)AB2698765(I)AB2472575(J)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2698745(K)AB2472585(L)AB2472585(L)AB2698745(K)AB2698745(K)AB2472595(M)AB2472605(N)AB2473365(O)AB2473395(P)AB2473395(P)1

ITS (type)
AJ234028——
AJ2339972—
AJ2339952——
AB2698775(j)AB2698775(j)AB2698775(j)AB2698775(j)AB2698785(k)AB2698785(k)AB2698775(j)AB2698775(j)AB2472385(l)AB2472385(l)AB2472385(l)AB2571155(m)AB2571155(m)AB2571155(m)AB2571155(m)AB2571155(m)AB2472395(n)AB2472395(n)AB2472395(n)AB2472395(n)AB2472395(n)AB2472405(o)AB2472415(p)AB2472385(l)AB2472385(l)AB2472435(q)AB2472435(q)AB2472435(q)1

atpB–rbcL (type)

Accession no.b

—
—
—
—
—
—
—
—
AB2706015(II)AB2706015(II)AB2706015(II)AB2706015(II)AB2706015(II)AB2706015(II)AB2706015(II)AB2706015(II)AB2472455(III)AB2471215(IV)AB2472455(III)AB2571225(V)AB2571225(V)AB2571225(V)AB2571225(V)AB2571225(V)AB2472465(VI)AB2472465(VI)AB2472475(VII)AB2472465(VI)AB2472465(VI)AB2472485(VII)AB2472465(VI)AB2472505(IX)
AB2472495(X)AB2472525(XI)
AB2472535(XII)AB2472535(XII)

trnK/matK (type)

Table 2. Tribal classification of cited species and their ITS, atpB–rbcL and trnK/matK sequences according to the DDBJ/EMBL/GenBank database

Taxonomy and Phylogeography of Nertera yamashitae
13

b

a

Spiradiclis bifida Kurz
Xanthophytum borneense (Valeton) Axelius
Xanthophytum capitellatum Ridl.
Amphidasya ambigua (Standl.) Standl.
Maschalocorymbus corymbosus (Blume) Bremek.
Pauridiantha paucinervis (Hiern) Bremek.
Pauridiantha symplocoides (S. Moore) Bremek.
Praravinia suberosa (Merr.) Bremek.
Pravinaria leucocarpa Bremek. Beaman
Urophyllum arboreum (Reinw. ex Blume) Korth.
Urophyllum ellipticum (Wight) Thwaites

Ophiorrhiza michelloides (Masam.) H.S. Lo
Ophiorrhiza pumila Champ. ex. Benth.

Ophiorrhiza kuroiwae Ohwi

Ophiorrhiza hayatana Masam.

Species
NKC003(RYU)
NKC005(RYU)
NKT0141(RYU)
NKT0142(RYU)
NKW001(RYU)
NKH001(RYU)
NKH002(RYU)
NKT0137(RYU)
GK3979(TNS)
J.B.H. 55(S)Axelius 316(S)
Ridsdale 2473(L)Clark & Watt 736(UPS)
Ridsdale 2471(L)Lantz 123(UPS)
Bremer 2090(UPS)—
Beaman 7950(S)Boeea 7887(S)Lundqvist 11085(UPS)

Voucher(Continued)

atpB–rbcL (type)AB2472435(q)AB2472435(q)AB2472445(r)AB2472445(r)AB2472425(s)AB2471475(t)AB2471475(t)AB2471475(t)AB2471475(t)—EU1455671
AJ2339962
EU1453371—
AJ2339982
EU1453381—
AJ2340012
DQ1317933
AJ2340022

ITS (type)AB2473375(Q)AB2473385(R)AB2473415(S)AB2473425(T)AB2473405(U)AB2472735(V)AB2472735(V)AB2472745(W)AB2472725(X)EU1453791
EU1453811
EU1453801
EU1453831
EU1453841
EU1453851
EU1453861
EU1453871
EU1453881—
EU1453891

Accession no.b

AB2472545(XIII)
AB2472555(XIV)
AB2472565(XV)
AB2472565(XV)
AB2472515(XVI)
AB2471505(XVII)
AB2471505(XVII)
AB2471505(XVII)
AB2471505(XVII)
—
—
—
—
—
—
—
—
—
—
—

trnK/matK (type)

Taxonomic treatment follows Bremer and Manen (2000).
Sequences in the DDBJ/EMBL/GenBank database; letters in parentheses indicate the types of three loci; references are as follows: 1 Rydin et al. (2009a, 2009b);
2
Bremer and Manen (2000); 3 Manen (GenBank unpublished data); 4 CL Anderson et al. (GenBank unpublished data); 5 Nakamura et al. (2006).

Urophylleae

Ophiorrhizeae

TribeaTable 2.

14
Goro Kokubugata et al.

Taxonomy and Phylogeography of Nertera yamashitae

15

Table 3. The top five accessions with high similarity to the sequences of Nertera yamashitae using BLAST andtheir localities
Species

LocusaLocality

Accession
no.b

Score
(bits)

Identities

659/684(96%)
658/684(96%)
657/684(96%)
656/684(95%)

ITS
Ophiorrhiza amamiana

JAPAN (Okinawa Is.)ITS(I)

AB269876

1144

Ophiorrhiza amamiana

JAPAN (Amami, Tokunoshima,& Okinawa Isls. in the Ryukyus)
TAIWANITS(H)

AB269875

1136

ITS(N)

AB247260

1128

JAPAN (Shikoku, Kyushu & Amami, ITS(K)
Tokunoshima, Okinawa, Ishigaki& Iriomote Isls. in the Ryukyus)
TAIWANITS(M)

AB269874

1120

AB2472591112

655/684(95%)

JAPAN (Tokunoshima &Okinawa Isls.)
TAIWAN

atpB–rbcL(m)AB2571151132

atpB–rbcL(q)AB247243

1132

Ophiorrhiza michelloides TAIWAN

atpB–rbcL(s)AB247242

1132

Ophiorrhiza japonica

JAPAN (Iriomote Is.); Taiwan

1132

Ophiorrhiza amamiana

JAPAN (Amami, Tokunoshima,& Okinawa Isls. in the Ryukyus)

atpB–rbcL(p, l) AB247241,
AB247238
atpB–rbcL(j)AB269877

1124

597/608(98%)
597/608(98%)
597/608(98%)
597/608(98%)
596/608(98%)

Ophiorrhiza michelloides TAIWAN

matK/trnK(XVI) AB247251

4220

Ophiorrhiza japonica

matK/trnK(VI)AB247246

4209
4205

Ophiorrhiza japonica

matK/trnK
AB247254,(XIII, XIV)
AB247255
JAPAN (Iriomote Is. in the Ryukyus) matK/trnK(VII) AB247248

4201

Ophiorrhiza hayatana

TAIWAN

4197

Ophiorrhiza japonica
Ophiorrhiza japonica
Ophiorrhiza japonica
atpB–rbcL
Ophiorrhiza japonica
Ophiorrhiza hayatana

matK/trnK

Ophiorrhiza hayatanaabJAPAN (Ishigaki & Iriomote Isls.
in the Ryukyus)
TAIWAN

matK/trnK(XI)

AB247252

2276/2335(97%)
2274/2334(97%)
2274/2335(97%)
2273/2334(97%)
2273/2335(97%)

Letters in parentheses indicate locus type in Table 2.
Sequences in the DDBJ/EMBL/GenBank database.

locus (2328 bp) of N. yamashitae had highest
similarity to 19 accessions of six Ophiorrhiza
species in Japan and Taiwan. No accession for
trnK/matK of Nertera species was found in the
DDBJ/EMBL/GenBank database.
Phylogenetic position of Nertera yamashitae at
the tribe level
In the MP analyses at tribe level based on the

ITS sequence (Fig. 2), 233 of the 313 variablecharacters were parsimony-informative, and 305
equally most parsimonious trees of 726 steps
were obtained, with a consistency index (CI)
0.65, a retention index (RI)0.87, and a rescaledconsistency index (RC)0.56. In the MP analyses at tribe level based on the atpB–rbcL sequences (Fig. 3), 110 of the 228 variable characters were parsimony-informative, and 216 equal-

16

Goro Kokubugata et al.

Fig. 2. Unrooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera
yamashitae in tribes Anthospermeae, Ophiorrhizeae, and Urophylleae based on the ITS locus. Species with
underlines and asterisks are members of tribes Ophiorrhizeae and Anthospermeae, respectively (Bremer andManen, 2000). Numerals above branches indicate bootstrap percentages (50%). Letters in parentheses
indicate loci types in N. yamashitae, other Nertera species, and Ophiorrhiza species (Tables 1 and 2).

ly most parsimonious trees of 288 steps were obtained (CI0.89, RI0.95, and RC0.85). Bothof the unrooted strict consensus trees based on
the ITS and atpB–rbcL loci indicated that N. yamashitae was not positioned in a clade with other
Nertera species nor in a clade of Anthospermeae(Figs. 2 and 3). On the other hand, N. yamashitae
was included in a clade with Ophiorrhiza species
and Spiradiclis bifida (tribe Ophiorrhizeae) basedon analysis of the ITS sequence (BP99.9%;
Fig. 2). Based on the atpB–rbcL sequence (Fig.
3), a clade consisting of N. yamashitae and Ophiorrhiza species had high bootstrap support(97.2%). In both of the unrooted strict consensus
trees, N. yamashitae included in a subclade withfour Ophiorrhiza species of O. amamiana(Hatus.) Koh Nakam., Denda, Kameshima et
Yokota, O. japonica Bl., O. hayatana Masam.
and O. michelloides (Masam.) H.S. Lo.
Phylogenetic relationship of Nertera yamashitae
to the other Ophiorrhiza species
To analyze the phylogenetic relationship within the subclade consisting of N. yamashitae andfour Ophiorrhiza species (Figs. 2 and 3), the MP
analysis was conducted based on the full set of
ITS, atpB–rbcL, and matK/trnK loci with otherfour Ophiorrhiza species, namely O. kuroiwae
Ohwi, O. pumila Champ. ex Benth., O. longiflora
Blume and an unknown Ophiorrhiza species as
an outgroup following the two independent
analyses based on ITS (Fig. 2) and atpB–rbcL

Taxonomy and Phylogeography of Nertera yamashitae

17

Fig. 3. Unrooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera
yamashitae in tribes Anthospermeae, Ophiorrhizeae, and Urophylleae based on the atpB–rbcL locus. Detail
explanation given in Fig. 2.

(Fig. 3) loci. In the MP analysis, 124 of 188
variable characters were parsimony-informative,
and six most parsimonious trees of 239 steps
were obtained, with CI0.86, RI0.94, andRC0.81 (Fig. 4).
The MP analysis (Fig. 4) indicated that N.
yamashitae was connected to a basal position of
a subclade consisting of O. amamiana, O. japonica, O. hayatana, and O. michelloides with highprobability (BP99.4%). Another dwarf leaf
species of O. michelloides in Taiwan was not sister to N. yamashitae.
Floral anatomy and morphology of Nertera
yamashitae
Flowers of N. yamashitae had a four-lobed andwhitish corolla, a style with stigma bilobed at the
top (Fig. 1) and four stamens (not shown). The
ovary of N. yamashitae was inferior, bilocular,
and had ca. 25 ovules in a locule, and the disk on
the ovary was thick (Fig. 5).

Discussion
Taxonomic reconsideration of Nertera yamashitae
Following previous taxonomic studies, thegenera Nertera and Ophiorrhiza commonly havea two-celled ovary, but the former has a single
ovule in a locule, while the latter has manyovules in a locule (e.g. Verdcourt, 1958; Liu andYang, 1998; Bremer and Manen, 2000). Furthermore, the former genus has an annular disk and astyle with stigma bilobed at the base, or no disk
(Puff, 1986), a four-lobed corolla, and four stamens; while the latter genus has a thick disk
(Fukuoka, 1979), a style with stigma bilobed at
the top, a five- or six-lobed corolla, and five or
six stamens (Chen and Zhu, unpubl. data). Although N. yamashitae was similar to other Nertera species in the numbers of corolla lobes andstamens, it was similar to Ophiorrhiza species in
the number of ovules, the morphologies of disk
and stigma. Out of these anatomical and morpho-

18

Goro Kokubugata et al.

Fig. 4. Rooted maximum parsimony, strict consensus tree to detect phylogenetic position of Nertera yamashitae
within the genus Ophiorrhiza based on the full dataset of ITS, atpB–rbcL, and trnK/matK loci. Numerals
above branches indicate bootstrap percentages (50%). Letters in parentheses indicate the types of three lociin N. yamashitae and Ophiorrhiza species (Tables 1 and 2).

logical characters, the number of ovules in an
ovary locule was thought to be an importantcharacter for delimitations at the tribes (Bremer
and Manen, 2000) and generic level (e.g. Hooker,
1873; Bremekamp, 1966) in the family Rubiaceae. In terms of anatomy and morphology, N.
yamashitae should not be treated as a species of
the genus Nertera.

Previous phylogenetic studies at tribal or
generic levels in the family Rubiaceae commonlyverified that tribe Anthospermeae including Nertera spp., and tribe Ophiorrhizeae including Ophiorrhiza spp., branched at a basal position in aclade of subfamily Rubioideae and that they were
obviously not sister groups (Bremer and Manen,
2000; Bremer, 2009; Rydin et al., 2009a, 2009b).

Taxonomy and Phylogeography of Nertera yamashitae

Fig. 5.

19

Anatomical and morphological observations of the disk and ovules in Nertera yamashitae. Bar500 m m.

The present molecular analyses reveal that N.
yamashitae is apparently included in the Ophiorrhiza clade in tribe Ophiorrhizeae but is distantly
related to the Nertera clade in tribe Anthospermeae.
For taxonomy based on the anatomical, morphological and molecular results, we propose totreat N. yamashitae as a species of the genus
Ophiorrhiza.
Ophiorrhiza yamashitae (T. Yamaz.) Kokubugata, Koh Nakam. et Yokota, comb. nov. Basionym: Nertera yamashitae T. Yamazaki, J. Jpn.
Bot. 73: 89–91, Figs. 1 and 2, 1998.
Fruit morphology is also a very valuable character to delineate the genera Nertera and Ophiorrhiza; the former has succulent (drupe) and thelatter has dry (dehiscent) fruits (e.g. Liu andYang 1998; Chen and Zhu, unpubl. data). However, no fruit of O. yamashitae has been observedin wild in spite of long-term efforts to find it(Yamazaki, 1998; Hotta, 2004), and thus thisspecies is assumed to have lost ability to reproduce by means of intrapopulation crossing(Hotta, 2004). Artificial hand-pollination between plants from different populations in culti-

vation could be useful to investigate the fruit
morphology of O. yamashitae.
Phylogeography of Ophiorrhiza yamashitae
In the present phylogenetic trees based on ITS,
atpB–rbcL, and trnK/matK (Fig. 4) O. yamashitae, being an insular-endemic to Amami
Island of the Ryukyus, was connected to a basal
position of a clade consisting of four Ophiorrhiza species, namely O. amamiana being endemic to the central Ryukyus (Nakamura et al.,2007); O. japonica widely distributing in China,
Japan, Taiwan, and Vietnam (Yamazaki, 1993);
and O. hayatana and O. michelloides being endemic to Taiwan (Liu and Yang, 1998; Nakamura
et al., 2006). Of four Ophiorrhiza species, O.
amamiana and O. japonica presently occur on
Amami Island (Hatusima and Amano, 1994).
However, our phylogenetic analyses suggest that
O. yamashitae is not derived from O. amamiana
nor O. japonica. These results imply that the ancestral species of O. yamashitae has been extinct,
and O. yamashitae could survived as a relict
species lineage in Amami Island.
Previously, some species lineages assumed tobe relicts are reported in the central part of the
Ryukyu Archipelago, including Amami Group20

Goro Kokubugata et al.

and Okinawa Group for terrestrial plants (Hatusima, 1975; Hotta, 2004), amphibians and reptiles (Hikida et al., 1989; Ota, 1998). Hikida andOta (1997) suggested that these relict species lineages of amphibians and reptiles migrated to thecentral Ryukyus when the area was connected tosurrounding landmasses by a landbridge via thenorthern and southern Ryukyus. After partial
submergence of the landbridge during the
Pliocene, the lineages were isolated in the central
Ryukyus and have been extinct in other areas ofthis archipelago (Hikida and Ota, 1997). There isa possibility that O. yamashitae might have asimilar phylogeographic background to the relict
species lineages of the terrestrial amphibians andreptiles in the central part of the Ryukyu Archipelago.
Parallel evolution and ecological aspects ofdwarf leaf species
In the Ophiorrhiza species in Japan and Taiwan, O. michelloides being endemic to Taiwan
was regarded as a dwarf leaf species (Masamune,
1932; Liu and Yang, 1998; Nakamura et al.,2006). Although O. yamashitae and O. michelloides commonly have dwarf leaves, they are distantly related in the present phylogenetic analyses. The results suggest that leaf dwarfism events
have occurred independently in O. yamashitae
and O. michelloides.
In habit environments, O. yamashitae is foundalong streams at low elevation on Amami Island(Yamazaki, 1998), while O. michelloides is foundat altitudes above 1,000 m in the Central Mountain Range of Taiwan (Liu and Yang, 1998).
However, they commonly occur on oligotrophic
rocky slopes (data not shown). In evolutionallyaspect of leaf dwarfism, Sugimoto (1957) suggested that oligotrophic environment is one ofpossible factors to lead to leaf dwarfism. We canhypothesize that leaf dwarfisms might be an
adaptation to survive in oligotrophic environments on the rocky slopes as a common phenomenon between O. yamashitae and O. michelloides, but parallel events in Amami Island andTaiwan. Further studies including soil physico-chemical investigations of these dwarf leaf
species on Amami Island and in Taiwan are necessary to address these hypotheses.
Acknowledgments
We thank Hiroshi Yamashita (Amami, Japan),
Hendrian (LIPI, Indonesia), Cheng-I Huang(HAST, Taiwan), Piyakaset Suksathan (Queen
Sirikit Bot. Gard., Thailand), and Monica
Suleiman (University of Malaysia, Sabah) for assisting on field trips. This study was conducted inconnection with the National Museum of Natureand Science projects on “Integration of Systematics and Molecular Phylogenetics in all Groupsof Organisms” and “Taxonomic and Conservation Studies on Endangered Plant Species inJapan”, and supported in part by Grants-in-Aid
for Scientific Research, Program C (JSPS; No.
20510220; GK) and the 21st Century COE program of the University of the Ryukyus (MY).
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