Bull. Natl. Mus. Nat. Sci., Ser. B, 47(1), pp. 45–50, February 22, 2021

Conﬁrmation of the Occurrence and Phylogenetic Backgroundsof Portulaca okinawensis var. amamiensis (Portulacaceae)
in Uke and Yoro Islands of the Ryukyus Archipelago
Goro Kokubugata1,*, Yumiko Hirayama1 and Masatsugu Yokota2
1

Department of Botany, National Museum of Nature and Science,4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Laboratory of Ecology and Systematics, Faculty of Science,
University of the Ryukyus, Nishihara, Okinawa 903–0213, Japan*E-mail: gkokubu@kahaku.go.jp(Received 9 November 2020; accepted 23 December 2020)

Abstract Occurrence of Portulaca okinawensis var. amamiensis is conﬁrmed from Uke and Yoro
Islands, which are islets of Amami Island in the Ryukyus. The ITS sequences of the plants of Kakeroma, Uke and Yoro Islands being geographically adjacent were identical, and were diﬀerent
from those of other islands in the central Ryukyus by having deletion of a nucleotide base pair.
This study is the ﬁrst to conﬁrm the occurrence of Portulaca okinawensis var. amamiensis in Yoro
Island.
Key words : Amami island group, ITS, Portulaca okinawensis, the Ryukyus.

Introduction
Portulaca okinawensis Walker et Tawada wasdescribed based on a type specimen collectedfrom Okinawa Island of the Ryukyu Archipelago(hereafter the Ryukyus; Fig. 1) in Japan (Walkerand Tawada, 1951). This species is endemically
distributed in the central Ryukyus, which arecomposed of the Amami and Okinawa islandgroups (Walker and Tawada, 1951; Hatusima,1975; Shinjo and Shinzato, 2006; Kokubugata etal., 2013, 2016; Fig. 1). This species is rarely
found on coastal rocky slopes, and is a criticallythreatened species on the Japanese red list (Ministry of Environment of Japan, 2020). Previously,
Kokubugata et al. (2013) distinguished plants ofP. okinawensis in Amami and Tokuno-shimaIslands of the Amami island group from those in
the Okinawa island group by the color of petals
and stems. They treated the former as an inde© 2021 National Museum of Nature and Science

pendent variety, namely P. okinawensis var. amamiensis Kokub., Koh Nakam. et Yokota, basedon a type specimen collected from Amami Island(Kokubugata et al., 2013). The two intraspeciﬁctaxa were supported by molecular phylogeneticanalyses using the internal transcribed spacer(ITS) region of nuclear ribosomal DNA(Kokubugata et al., 2013, 2016).
In 2017, we had a ﬁeld survey of Uke Island
and Yoro Island, which are adjacent to Kakeroma
Island of the Amami island group (Fig. 1), andfound a population of plants that were identiﬁed
as P. okinawensis by their morphological characters on the two islands. The objective of thisstudy was to determine the taxonomic status ofthese plants collected from Uke and Yoro Islands
based on morphological comparisons, and to elucidate their phylogenetic relationships with thosefrom other islands in the central Ryukyus using
molecular phylogenetic analysis of the ITS
region of nuclear DNA.

46

Goro Kokubugata et al.

Fig. 1. Map of Uke and Yoro Islands and adjacent areas. Solid circles indicate the locations of plants investigatedon Amami, Kakeroma, Uke, and Yoro Islands.

Materials and Methods
Field surveys and plant materials
We conducted a ﬁeld survey of Uke Island on
September 18 and Yoro Island on September 20,
2017. Two individuals of plants identiﬁed as P.
okinawensis were collected from each populationof both islands (GK20228 and 20229 from Uke
Island; GK20305 and 20308 from Yoro Island)for detailed taxonomic analysis following themethod of Kokubugata et al. (2013) and molecular phylogenetic analysis using the ITS region.
Voucher specimens for the present study weredeposited in the herbarium of National Museumof Nature and Science, Japan (TNS).
DNA extraction, PCR, and sequencing
Genomic DNA was extracted from silica gel-dried leaves using the DNeasy Plant Mini Kit(Qiagen, Valencia, CA, USA), following themanufacturerʼs protocol. The ITS region ofnuclear ribosomal DNA was ampliﬁed pantropic
by polymerase chain reaction (PCR) using aniCycler (Bio-Rad, Hercules, CA, USA) and theforward primer AB101 (5′-ACG AAT TCA AGG
TCC GGT GAA TGT TTC G-3′) and reverse
primer AB102 (5′-TAG AAT TCC CCG GTTCGC TCG CCG TTA C-3′) (Douzery et al.,1999). Ampliﬁcations were performed using
TaKaRa Sapphire Amp Fast Master Mix(TaKaRa, Otsu, Japan) with Sapphire Amp(TaKaRa). The PCR proﬁle was 35 cycles for 5 sat 94°C, 5 s at 50°C, and 5 s at 72°C after an initial denaturation step for 3 min at 94°C. PCR
products were checked by electrophoresis before
puriﬁcation using the Illustra ExoProStar (GEPortulaca okinawensis var. amamiensis in Uke and Yoro Islands
Table 1.

47

GenBank accession numbers of the ITS sequences of Portulaca plants used for phylogenetic analysis
Taxon

IngroupP. okinawensis var. okinawensis

var. amamiensis

Outgroup
P. psammotropha

Collection locality

Collection
number(herbarium)

Japan, central Ryukyus, Okinawa island group,
Okinawa Isl., Onna.
Japan, central Ryukyus, Okinawa island group,
Tonaki Isl.
Japan, central Ryukyus, Amami island group,
Amami Isl., Amami.
Japan, central Ryukyus, Amami island group,
Kakeroma Isl., Akitoku.
Japan, central Ryukyus, Amami island group,
Tokuno-shima Isl., Amagi.

G. Kokubugata
12873 (TNS)
G. Kokubugata
17300 (TNS)
G. Kokubugata
15434 (TNS)
G. Kokubugata
18970 (TNS)
G. Kokubugata
12141 (TNS)Japan, Bonin, Chichi-jima Isl.

G. Kokubugata
13338 (TNS)
G. Kokubugata
13338 (TNS)

Taiwan, Pingtung, Liuqiu Shiang.

GenBank
accession no.*AB823837a
AB823845a
AB823823a
LC133271b
AB823831a
LC018836c
AB823850a* ITS sequences registered in the DNA Data Bank of Japan (DDBJ): aKokubugata et al. (2013); bKokubugata et al.
(2016); cKokubugata et al. (2015).

Healthcare, Tokyo, Japan). The cycle sequencing
reaction was performed using the BigDye™ Terminator Cycle Sequencing Kit ver. 3.1 (AppliedBiosystems, Foster City, CA, USA). Primers
used for sequencing were AB101 and AB102.
The Sanger sequencing products were then puriﬁed by ethanol precipitation. Automatedsequencing was performed on the 3130xl GeneticAnalyzer (Applied Biosystems). The electropherograms were analyzed using ATGC ver. 4.01
(Genetyx Co., Tokyo, Japan). Sequence datafrom this study were deposited in the DNA Data
Bank of Japan (DDBJ) (LC593637 andLC593638 for GK20228 and 20229 from Uke
Island; LC593639 and LC593640 for GK20305
and 20306 from Yoro Island, respectively).
ITS data from the DNA database
Kokubugata et al. (2013, 2016) reported ﬁve
ITS types of P. okinawensis: two types of P. okinawensis var. okinawensis from Okinawa andTonaki Islands in the Okinawa island group, andthree types from Amami, Kakeroma, andTokuno-shima Islands in the Amami islandgroup. To elucidate the phylogenetic relationships among the plants of P. okinawensis on Ukeand Yoro Islands and those on other islands ofthe central Ryukyus, the ﬁve ITS types of P. okinawensis previously provided by Kokubugata etal. (2013, 2016) were subjected to the present
molecular analyses as ingroup members (Table1). We used P. psammotropha Hance from Bonin
Island of Japan and Taiwan as outgroup members(Table 1) following Kokubugata et al. (2015).
Finally, eleven operational taxonomic units(OTUs), nine ingroup and two outgroup members, were obtained for the present phylogeneticanalysis.
Phylogenetic analyses
DNA sequences were aligned using ClustalW
1.8 (Thompson et al., 1994) and then adjusted
manually. Phylogenetic analyses were performed
based on a maximum parsimony (MP) criterion,
using PAUP* version 4.0b10 (Swoﬀord, 2002).
For MP phylogenetic analysis, indels weretreated as missing data, characters were treatedas unordered, character transformations wereweighted equally. The branch collapse option
was set to collapse at a minimum length of zero.
A heuristic parsimony search was performed
using 200 replicates of random additions ofsequences, with ACCTRAN character optimization, tree bisection–reconnection (TBR) branch

48

Goro Kokubugata et al.

Fig. 2. Portulaca okinawensis and its habitats on Uke and Yoro Islands. A, B. Habitat. C, D. Plant. A, C. Uke
Island. B, D. Yoro Island.

swapping, and MULTREES and STEEPESTDESCENT options on. Statistical analysis ofeach clade was performed using bootstrap analysis (Felsenstein, 1985). One thousand replicatesof heuristic searches, with the TBR branch swapping switched on and MULTREES options oﬀ,
were performed to calculate bootstrap values
with 10,000 repeats. The MP tree was generatedusing TreeView (Page, 1996).
Results
Populations of plants identiﬁed as P. okinawensis were found on dry and sunny seaside rocky
slopes with typical seaside plants in the Ryukyus
such as Hedyotis strigulosa (Bartl. ex DC.) Fosberg var. parvifolia (Hook. et Arn.) T.Yamaz.
(Rubiaceae) on Uke and Yoro Islands (Fig. 1A,
B). On both islands, these plants had light-green
stems and light-yellow petals (Fig. 1C, D).
The ITS sequences of the plants from Uke andYoro Islands were identical to that of P. okinaw-ensis var. amamiensis from Kakeroma Island.
The ITS sequences of the plants from the threeislands were diﬀerent from those of the plantsfrom other islands of the central Ryukyus byhaving a deletion of adenine inserted at base pair
173 in the ITS sequences.
After aligning the ITS sequences from the 11OTUs, a matrix of 598 bp was obtained. In the MP
analysis, 11 of the 15 variable characters were parsimony-informative, and a single most parsimonious tree of 15 steps was obtained (Fig. 3), with aconsistency index of 1.000, retention index of1.000, and rescaled consistency index of 1.000.
The MP tree showed two major clades in theingroup (Clades I and II in Fig. 3). Clade I consisted of three OTUs of P. okinawensis var. amamiensis from Amami, Tokuno-shima and Kakeroma Islands; and four OTUs of P. okinawensis
from Uke and Yoro Islands evaluated herein.
Clade II consisted of two OTUs of P. okinawensis var. okinawensis from Okinawa and TonakiIslands.

Portulaca okinawensis var. amamiensis in Uke and Yoro Islands49

Fig. 3. Maximum parsimonious tree for plants of Portulaca okinawensis collected from the central Ryukyus
based on ITS sequences. Numbers above branches indicate bootstrap percentages (lower, ≤50%). Bold indicates plants of P. okinawensis collected from Uke and Yoro Islands.

Discussion
As mentioned brieﬂy, Kokubugata et al. (2013,2016) reported that two varieties of Portulaca okinawensis were distinguishable according to stemand petal color: P. okinawensis var. okinawensis
distributed in the Okinawa island group had reddish green stems and dark yellow petals, whereas
P. okinawensis var. amamiensis distributed in theAmami, Kakeroma and Tokuno-shima Islands ofthe Amami island group had light green stems andlemon-colored petals. The present morphologicalcomparison taxonomically concludes that plants
collected from Uke and Yoro Islands should betreated as P. okinawensis var. amamiensis. The
present MP analysis of the ITS sequences supports
this morphological conclusion.
In the Amami island group, this variety has
been reported to occur on Amami, Kakeroma andTokuno-shima Islands by Kokubugata et al.
(2013, 2016), and on and Uke Islands by Shinjoand Shinzato (2006; as P. okinawensis). This
study is the ﬁrst to report the occurrence of P.
okinawensis var. amamiensis on Yoro Island.
It is noteworthy that the plants from Kakeroma, Uke and Yoro Islands share the same ITS
sequences. The three islands are geographicallyadjacent, being separated from each other by lessthan 5 km, and have similar ﬂora (Suzuki et al.,2020). The consistency of the ITS sequencesamong the three islands must be resulted fromtheir geographical relationships. On the otherhand, it is unexpected that the ITS sequences ofthe plants from the three islands are diﬀerent
from those from Amami Island. Previously, any
speciﬁc biological barrier has been unknown
between the three islands and Amami Island. The
present study did not reveal phylogeographic
relationship of the plants from Kakeroma, Uke
and Yoro Islands with those from other islands of
the Amami island group in detail, because indels
on the ITS sequences were treated as missing
data in the present MP analysis following
Kokubugata et al. (2013, 2016). Further molecular phylogenetic analyses using other DNA
sequences, including cpDNA, are required to better understand the phylogenetic relationships ofP. okinawensis var. amamiensis.
Acknowledgments
We thank M. Tabata for providing valuable
information, H. Sannokyo (Minorino-kai in Uke
Island) and Setouchi Town Oﬃce for assistingﬁeld trip in Uke and Yoro Islands, and R.
Matsuda for assisting DNA experiments. This
study was partly funded by a Grant-in-Aid for
Scientiﬁc Research (B) (JSPS KAKENHI Grant
Number 25290085).

50

Goro Kokubugata et al.

References
Douzery, E. J. P., Pridgeon, A. M., Kores, P., Linder, H.
P., Kurzweil H. and Chase, M. W. 1999. Molecularphylogenetics of Diseae (Orchidaceae): A contribution
from nuclear ribosomal ITS sequences. American Journal of Botany 86: 887–899.
Felsenstein, J. 1985. Conﬁdence limits on phylogenies:
An approach using the bootstrap. Evolution 39: 783–791.
Hatusima, S. 1975. Flora of the Ryukyus, Added and Corrected. Okinawa Association of Biology Education,
Naha (in Japanese).
Kokubugata, G., Ito, T. and Yokota, M. 2016. Conﬁrmation of the occurrence of Portulaca okinawensis var.
amamiensis (Portulacaceae) in Kakeroma Island of theRyukyus Archipelago, Japan using morphological andmolecular data. Bulletin of the National Museum ofNature and Science, Series B 42: 67–71.
Kokubugata, G., Nakamura, K., Hirayama, Y. and Yokota,
M. 2013. Taxonomic reconsideration of Portulaca okinawensis (Portulacaceae) in the Ryukyus Archipelago
based on morphological and molecular data. Phytotaxa
117: 11–22.
Kokubugata, G., Kato, H., Iamonico, D., Umemoto, H.,
Ito, T., Nakamura, K., Murakami, N. and Yokota, M.
2015. Taxonomic reexamination of Portulaca boninen-sis (Portulacaceae) in the Bonin (Ogasawara) Islands ofJapan using molecular and morphological data. Phytotaxa 217: 279–287.
Ministry of Environment of Japan. 2020. Red List ofJapan. http://www.env.go.jp/press/ﬁles/jp/114457.pdf(in Japanese) [Accessed 2 November 2020].
Page, R. D. M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in Bioscience 12: 357–358.
Shinjo K. and Shinzato, T. 2006. Portulaca okinawensis
Walker et Tawada. In: Okinawa Prefecture (ed.), Red
Data Okinawa, revised version, p. 72. Okinawa Prefecture, Naha (in Japanese).
Suzuki, E. 2020. Flora on Kakeroma, Uke, and Yoro
Islands. Occasional Papers 61: 32–33 (in Japanese).
Swoﬀord, D. L. 2002. PAUP: Phylogenetic analysis usingparsimony, version 4.0b10. Sinauer Associates, Sunderland, Massachusetts.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. 1994.
CLUSTAL W: Improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position speciﬁc gap penalties and weight matrix
choice. Nucleic Acids Research 22: 4673–4680.
Walker, E. H. and Tawada, S. 1951. A new species of Portulaca from Okinawa. Journal of the Washington Academy of Sciences 41: 138.