JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 90, No. 4, 447-452. 2000

Origin of Fan Palm (Livistona chit-zensis R. Br . var. subglobosa Becc.) in Aoshima, Japan

NAOTO YOSHIDA,‘* RIKA NOBE,’ TAKASHI YAMADA, KIHACHIRO OGAWA,’ AND YOSHIKATSU MUROOKA3

Department of Biochemistry and Apphed Biosciences, Faculty ofAgriculture, Miyazaki University, I-I Gakuen Kibanadai-Nishi, Miyazaki-shi 889-2192, l Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,

Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527,2 and Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565-0871,3 Japan

Received 15 November 1999/Accepted 24 July 2000

RAPD and RFLP analyses were carried out to determine the origin of Livistona chinensis R. Br. var. sub- globosa Becc. from Iriomotejima, Ishigakijima, Okinawa, Yakushima, Tanegashima, Cape Sata, Cape Toi, Tsukishima, and Aoshima, Japan. RAPD data obtained using 5 random primers were clustered using UPGMA or the neighbor-joining method. Each population was classified into three clusters based on the phylogenetic tree. L. chinensis plants from Yakushima, Tanegashima and Cape Sata belonged to the isologous cluster, and those from Ishigakijima and Okinawa are contained in a different cluster. L. chinensis from Okinawa and Ishigakijima could be differentiated by being the oldest based on the genetic distance. The area that covers Ishigakijima and Okinawa is thought to be the origin of L. chinensis. L. chinensis plants from Iriomotejima were contained in the same cluster as those from Aoshima. The phyiogenetic trees constructed by both RAPD and RFLP analyses indicate the possibility that seeds or green woods of L. chinens& were dispersed by tidal current from the south field around Iriomotejima, and they were washed to Aoshima and established gradually. Therefore, we support the drifting-ashore-naturalized-plant hypothesis on the origin of L. chinensis in Aoshima.

[Key words: Livistona chinensis R. Br. var. subglobosa Becc., Arecaceae, RAPD, RFLP, fan palm]

Livistona (Arecaceae) comprises 28 species and is dis- tributed in Australia, New Guinea, Southeast Asia such as the Philippines, and South China. Livistona chinensis R. Br. var. subglobosa Becc. or Livistona japonica Nakai (Fig. l), which is known as “fan palm”, is found in Taiwan, the islands of Okinawa and as far north as Kyushu, Japan, where it grows along shores washed by the warm Kuroshio ocean current. However, virgin L. chinensis forest is now found only in the islets of Ao- shima and Tsukishima in Miyazaki Prefecture, Kyushu. Aoshima is the extreme northern limit of the Arecaceae plant community. The entire islet (4.4 ha) is covered with about 4000 L. chinensis plants, giving it the appearance of a subtropical jungle. L. chinensis stands are rare, even though the plant can be found throughout the re- gion including the Okinawa islands. Because there are few natural L. chinensis forests remaining elsewhere, it is of interest why the palm grows abundantly only in Aoshima. There are conflicting hypotheses about the ori- gin of L. chinensis in Aoshima. One hypothesis is that seeds or green woods of L. chinensis were carried north- ward by Kuroshio ocean current from Taiwan or Oki- nawa to Aoshima, where the plant established itself, become naturalized, and eventually grew into a thick forest (drifting-ashore-naturalized-plant hypothesis) (1). The other hypothesis is that the plant flourished through- out the region when the climate was warm but after the tertiary period, changes in the climatic zones led to its gradual death, remaining only on islets washed by a moderately warm sea (relic hypothesis) (2). Although the former hypothesis appears to have more credence among the general public, it is not based on rigorous scientific

* Corresponding author.

evidence. These two hypotheses were reported in 1918 and 1925, respectively, in which the authors relied on a combination of guesswork, well-known facts, and old ob- servation records.

Recently, PCR-based molecular markers have been used for analysis of genetic variation in natural popula- tions (3-5). One such technique, namely, random am- plified polymorphic DNA (RAPD) (6) uses arbitrary pri- mers, and provides a convenient and rapid assessment of differences in the genetic composition of the related in- dividuals (7). By utilizing new scientific findings and DNA analysis techniques, we provide here a new inter-

FIG. 1. L. chinensis R. Br. var. subglobosa Becc. in Aoshima islet, Miyazaki Prefecture. The main trunk is fat, with a wavy, circu- lar pattern.

447

448 YOSHIDA ET AL. .I. BIOSCI. BIOENG.,

Pacific ocean

FIG. 2. Nine locations where L. chinensis leaves were collect are shown in the map of Southern Japan.

pretation of the origin and spread of L. chinensis based on analysis of the DNA polymorphism using RAPD and restriction fragment length polymorphism (RFLP) mark- ers.

MATERIALS AND METHODS Plant material and DNA extraction Leaves of five

L. chinensis plants were collected from several locations in natural forests of Iriomotejima, Ishigakijima, Oki- nawa, Yakushima, Tanegashima, Cape Sata, Cape Toi, Tsukishima, and Aoshima as shown in Fig. 2. Natural L. chinensis stands no longer exist in Taiwan. Young and fully expanded leaves were stored at -80°C until their total DNA was extracted. Frozen leaves in liquid nitrogen were powdered using a mortar and pestle and DNA extraction was carried out using the DNA extrac- tion kit obtained from Nippon Gene Inc. Crude DNA was precipitated using ethanol, dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), and puri- fied using a minicolumn purchased from Omega Biotek Inc., GA, USA.

RAPD analysis Five dodecamer oligonucleotides, A22 (5’-GCCTGCCTCACG-3’), A23 (5’-ACTGACCT AGTT-3’), A27 (5’-ATCGCGGAATAT-3’), A30 (5’-GAC CTGCGATCT-3’), and A52 (5’-CTTGTCATGTGT-3’) (Nippon Gene, Toyama), which produced polymorphic and reproducible RAPD bands, were used as the pri- mers. Reaction mixtures containing 1 x PCR buffer (Toyobo, Tokyo), 1.5mM MgC12, 200 PM dNTP, 0.5 PM primer, 80 ng of template DNA, and 2.5 U Tuq DNA polymerase (Toyobo) were overlaid in a tube. Am- plification was performed using a Perkin-Elmer 2400 thermal cycler programmed as follows: one cycle at 95°C for 3min, followed by 40 cycles of 93°C for 1 min, 40°C for 2 min, and 72°C for 2 min, followed by one cycle at 72°C for 5 min. The amplified mixtures maintained at 4°C were electrophoresed in 1.8% agarose gel. The gel was stained with ethidium bromide and the presence or absence of each band was recorded. Am- plification was performed at least three times, and reproducible data were retained.

RFLP analysis Plasmid (pRNE101) with a fragment

coding for rRNA derived from Chlorella ellipsoidea C-87 (8) was digested by both SaA and HindIII. The gel-purified rRNA gene (about 2.7 kbp) fragment coding small subunit rRNA was labeled using digoxigenin (DIG)-ll-dUTP by the random primer method using a DIG DNA labeling and detection kit (Boehringer Mann- heim GmbH, Mannheim, Germany). The DIG-labeled rRNA fragment was used as the probe for RFLP analy- sis. The genomic DNA of L. chinensis was digested by BarnHI, EcoRI, HindIII, or &I, and the resulting frag- ments were separated by electrophoresis on a 1.0% (w/v) agarose gel. The DNA was blotted from the gel onto a positively charged nylon membrane (Hybond- N-t ; Amersham International PLC, Amersham, UK) by capillary transfer using 0.4 M NaOH as the transfer solu- tion. Membrane blots were hybridized using the probe overnight at 68°C in a buffer (pH 7.2) containing 0.5 M Na2HP04, 1 mM EDTA and 7% SDS. The membranes were washed using a buffer (pH 7.2) containing 40mM Na2HP04 and 1% SDS at 68°C. Hybridized DIG-labeled probes on the membranes were detected calorimetrically using an alkaline-phosphatase-labeled antibody to DIG using the above-mentioned kit (Boehringer).

Data analysis Nucleon type was determined from the band profile of RAPD or RFLP. Gene frequency in each population was determined from the nucleon type. Genetic distance was calculated from these values in the same way as the procedure that seeks genetic distance from the heterozygotic degree or genetic diversity used in the study of the protein polymorphism (9). Genetic iden- tity (0 can be estimated as described by Nei and Li (9) as follows:

where Jxi and Jti are the frequency of the ith DNA frag- ment detected in populations X and Y, respectively. Since RAPDs and RFLPs are dominant in diploid popu- lation, J, and Jr must be estimated. The genetic distance (0) between populations was computed as follows:

D= -1ogeZ Phylogenetic trees were constructed based on the calculat- ed genetic distance using both unweighted pair group method arithmetic (UPGMA) (10) and the neighbor-join- ing method (1 l), as implemented in the NEIGHBOR pro- gram of the PHYLIP software package (12).

RESULTS RAPD and RFLP analyses Five random primers

A22, 23, 27, 30, and 52 were used to amplify 7, 6, 8, 11, and 8 DNA bands, respectively by polymerase chain reac- tion (PCR). The bands common to all plants in the de- tected amplified DNA band were 11. The eight RAPD markers amplified by using A27 are shown in Fig. 3. There were two to five out of eight detected marker bands in five L. chinensis plants obtained from Aoshima.

Seven marker bands were detected by RFLP analysis of BarnHI-digested DNAs of L. chinensis. Genetic poly- morphism was not detected by RFLP analysis of EcoRI, HindIII, and P&I-digested DNAs. Figure 4 shows the genetic polymorphism of five L. chinensis plants ob- tained from Aoshima, as an example. In the seven de- tected RFLP marker bands, the marker band indicated by the third arrowhead from the bottom was not de- tected in plants obtained from Aoshima.

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A B C ,D, E 1

FIG. 3. Example of RAPD analysis of L. chinensis using A27 as the primer. A27 amplified a total of eight DNA as bands, indicated by arrowheads. Lane alphabetical characters correspond to a plant from each population in the five geographic areas: Iriomotejima (A), Ishigakijima (B), Tsukishima (C), Cape Toi (D), and Aoshima (E).

21.23- kbp

4.15 - 4.27- 3.52 -

FIG. 4. Example of RFLP analysis of BumHI-digested DNAs from five L. chinensis plants from Aoshima using rRNA gene of C. ellipsoidea C-87 as the probe.

ORIGIN OF L. CHINENSIS 449

0 A22 A23 A27 A30 A52

Primer

J

FIG. 5. Nucleon divergence among nine geographic areas for each of five kinds of random primers. Nucleon divergence (d,) ob- tained by RAPD analysis for each primer compared. The nine popu- lations are as follows: 0, Iriomotejima; 0, Ishigakijima; A, Oki- nawa; A, Tanegashima; 0, Yakushima; n , Cape Sam, 0, Tsukishima; +, Cape Toi; 0, Aoshima.

Nucleon divergence RAPD in other words, a nu- cleomorph is shown in Fig. 5. When A27 was used as the primer, nucleon divergence in each population was the highest, i.e., 50-75%. On the other hand when A23 primer was used, nucleon divergence in each population was the lowest, i.e., O-25%. When genes were amplified by A52, nucleon divergence was intermediate at 40%. When A22 and A30 were used, the dispersion of the nucleon divergence was large, and it was 17-65x and 17-63x, respectively. Therefore, nucleon divergence depends on the primer used. Because the genetic distance is calculated based on the mean gene frequency, phylogenetic analysis by RAPD is a suitable method. In contrast to the fact that the divergence of the human mitochondrial DNA profile by geographic area is less than 1% (13), that of L. chinensis was as high as 33- 51% in the nucleon divergence within the population. Though a phenotype change was not recognized, many neutral mutations occurred in the plant (14, 15).

Nucleon type comparison Table 1 shows three mea- sures of nucleon divergence within and between the nine populations examined based on RAPD analysis. These measures are related to each another by Eq. 1:

TABLE 1. Nucleon divergence based on RAPD within and between nine L. chinensis R. Br. var. subglobosa Becc. populations

Iriomotejima Ishigakijima Okinawa Tanegashima Yakushima Cape Sata Tsukishima Cape Toi Aoshima Iriomotejima 0.4318 0.0739 0.1439 0.0991 0.0932 0.1214 0.0524 0.0483 0.0423

Ishigakijima 0.4943 0.4091 0.0985 0.1287 0.1227 0.1554 0.1409 0.0824 0.1219

Okinawa 0.6136 0.5568 0.5076 0.1097 0.1122 0.1289 0.1432 0.1070 0.1400

Tanegashima 0.5409 0.5591 0.5894 0.4518 0.0119 0.0450 0.0814 0.0690 0.0682

Yakushima 0.5295 0.5477 0.5864 0.4582 0.4409 0.0404 0.0805 0.0654 0.0610

Cape Sata 0.5000 0.5227 0.5454 0.4336 0.4236 0.3255 0.1100 0.0958 0.1114

Tsukishima 0.4955 0.5727 0.6242 0.5345 0.5282 0.5000 0.4545 0.0449 0.0414

Cape Toi 0.5057 0.5284 0.6023 0.5364 0.5273 0.5000 0.5136 0.4830 0.0349

Aoshima 0.4841 0.5523 0.6197 0.5200 0.5073 0.5000 0.4945 0.5023 0.4518

The divergence is calculated using the published method (20). The mean pairwise divergence between individuals (n = 5) within populations (dJ appears on the diagonal Values below the diagonal (dxy) are the mean pairwise divergences between individuals belonging to two different populations, X and Y. Values above the diagonal (d) are interpopulation divergences, corrected for variation within these populations using Eq. 1.

450 YOSHIDA ET AL. J. BIOSCI. BIOENF.,

TABLE 2. Matrix of genetic distances based on frequencies of RAPD markers among nine populations -.

Iriomotejima Ishigakijima Okinawa Tanegashima Yakushima Cape Sata Tsukishima Cape Toi Aoshima

Iriomotejima - Ishigakijima Okinawa Tanegashima Y akushima Cape Sata Tsukishima Cape Toi Aoshima

0.1362 0.3141 0.1953 0.1807 0.2136 0.0984 0.0921 0.0786 0.1965 0.2553 0.2391 0.2797 0.2843 0.1587 0.2400

0.2353 0.2378 0.2373 0.3215 0.2379 0.3120 - 0.0314 0.0711 0.1612 0.1382 0.1329

- 0.0634 0.1574 0.1287 0.1165 - 0.1932 0.1664 0.1975

- 0.0879 0.0787 - 0.0673

-

d=d,,-0.5 (d,fd,) (1) where d, is the mean pairwise divergence (%) between in- dividuals within a single population (X), dy is the cor- responding value of another population (Y), dxy is the mean pairwise divergence between individuals belonging to two different populations (X and Y), and d is a mea- sure of the interpopulation divergence corrected for in- trapopulation divergence. Okinawa as a group is more variable (d,=O.5076) than other groups. Cape Sata shows nearly identical amounts of within-group varia- tion (d,=O.3255). When the interpopulational distances (dxy) are corrected for intrapopulation variation, they become very small (d=0.0349-0.1554). The mean value of corrected distances among populations (d=0.0957) is about one-fifth of the mean distance between individuals within a population (0.4341). Most of the nucleon varia- tion in the L. chine&s is therefore shared between popu- lations. The mean value of corrected distances among hu- man populations, such as Caucasian and Negro, is one- tenth of the mean distance between individuals within a population (16). These results suggest that only neutral type of mutation occurs in L. chinensis.

Evolutionary tree Genetic distances between popu- lations calculated based on RAPD analysis in accordance with the procedure of Nei and Li (9) are shown in Table 2. The shortest genetic distance was 0.0314 between Yakushima and Tanegashima L. chinensis plants. A phylogenetic tree was constructed based on the genetic distance obtained by UPGMA, and is shown in Fig. 6A. Clusters were formed among L. chinensis populations in Okinawa and Ishigakijima, among those in Iriomote- jima, Tsukishima, Cape Toi, and Aoshima, and among those in Tanegashima, Yakushima, and Cape Sata. The genetic distances between Aoshima and Cape Toi was 0.0672. The genetic distance between the cluster (Ao- shima, Cape Toi) and Tsukishima, and between the clus- ter (Aoshima, Cape Toi, Tsukishima) and Iriomotejima were 0.0834 and 0.0898, respectively. An unexpected result was that the very distant Iriomotejima was con-

A 0.0777 o.0337 Cape Sata 00’57 Yakushima

Tanegashima 0.1246 0.0336

0.0417 Aoshima

G 0.0449 Cape Toi

Tsukishima lriomotejima

0.0983 lshigakijima

Okinawa

0.05

B / Okinawa

lshigakijima

4 Cape Sata

Cape Toi

Iriomotejimaq’Tsukishima

Aoshima 0.05 FIG. 6. Phylogenetic tree constructed using UPGMA (A) and the

neighbor-joining method (B) based on the interpopulation genetic distance obtained by RAPD analysis. Numbers indicate the value of genetic distance moiety. The bar shows the genetic distance.

tamed the cluster with Aoshima. Genetic distances among the nine L. chinensis popula-

tions were calculated as described above and used to

TABLE 3. Matrix of genetic distances based on frequencies of RFLP markers among nine populations

Iriomotejima Ishigakijima Okinawa Tanegashima Yakushima Cape Sata Tsukishima Cape Toi Aoshima

Iriomotejima Ishigakijima Okinawa Tanegashima Yakushima Cape Sata Tsukishima Cape Toi Aoshima

- 0.1711 0.2193 0.1711 0.0619 0.0771 0.0799 0.0657 0.0417 - 0.0891 0.1823 0.0951 0.0429 0.0912 0.1823 0.0749

- 0.2967 0.2096 0.1424 0.2056 0.2967 0.1589 - 0.095 1 0.1707 0.0912 0.1823 0.1157

- 0.0836 0.0040 0.0559 0.0285 - 0.0796 0.1707 0.0364

- 0.0912 0.0246 - 0.1157

-

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A 0.0355

I- lriomotejima

Tsukishima

Aoshima

Cape Toi

Cape Sata

lshigakijima

Tanegashimz

Okinawa

Tanegashima

Cape Sata

c

Aoshima

Ishigakijimal Okinawa

0.05 FIG. 7. Phylogenetic tree constructed using UPGMA (A) and

neighbor-joining method (B) based on the interpopulation genetic distance obtained by RFLP analysis. Numbers indicate the value of genetic distance moiety. The bar shows the genetic distance.

construct a phylogenetic tree by the neighbor-joining method, as shown in Fig. 6B. L. chinensis plants from Tanegashima, Cape Sata, and Yakushima belong to one group from the unrooted phylogenetic tree. L. chinensis plants from Cape Toi, Aoshima, and Tsukishima belong to another group. L. chinensis plants from Aoshima are genetically close relatives of those from Iriomotejima as also shown by UPGMA analysis. L. chinensis plants from Ishigakijima and those from Okinawa are genetical- ly distant from the above two groups. In particular, L. chinensis plants from Okinawa are genetically distant from all other group.

Table 3 shows the genetic distances based on RFLP analysis among the nine populations. The shortest genet- ic distance was 0.0040 between Yakushima and that from Tsukishima. Phylogenetic trees shown in Figs. 7A and B were constructed in accordance with UPGMA and the neighbor-joining method, respectively. The phylogenetic trees were slightly different from those in Figs. 6A and B. RFLP analysis was carried out using only one kind of restriction enzyme (BumHI) and DNA polymorphism was observed. If RFLP were carried out using multiple restriction enzymes, the result would ap- proach that of RAPD analysis. In RFLP analysis, the constructed phylogenetic tree using UPGMA and the neighbor-joining method shows that the genetic distance was small between L. chinensis from Aoshima and that from Iriomotejima (0.0710), and large between that

ORIGIN OF L. CHINENSIS 451

from Okinawa and other populations (0.2022). These re- sults correspond to those of RAPD analysis.

DISCUSSION

In the RFLP analysis, only one restriction enzyme (BarnHI) is used. On the other hand, five primers were used in RAPD analysis. Thus, the reliability of RAPD analysis is higher than that of RFLP analysis. When nucleon types from the amplified DNA band profiles (RAPD analysis) were compared, the nucleon diversity among populations in each area was found to be very high, with a mean of 43.4%. Judging from the phylogenetic tree constructed based on the genetic dis- tances (Fig. 6A), the earliest L. chinensis flourished in the area around the islands of Ishigakijima and Oki- nawa. In other words, the results suggest that this area is the origin of L. chinensis. Furthermore, it is assumed from the phylogenetic tree (Fig. 6A) that L. chinensis spread from Okinawa and Ishigakijima northward to Tanegashima and Yakushima, and southward to Iriomotejima. The possibility that the area around Okinawa is cradle of L. chinensis is high, because L. chinensis from Okinawa is more genetically distant from the other groups than L. chinensis from Ishigakijima based on the unrooted phylogenetic tree constructed using the neighbor-joining method (Fig. 6B). The fact that L. chinensis from Aoshima is proximal in terms of genetic distance with that from Iriomotejima suggests that seeds or green woods of L. chinensis were carried to Aoshima by the Kuroshio ocean current, where they gradually became established and flourished. This our findings support the hypothesis that the plant drifted ashore on Aoshima and became naturalized.

A genetic distance of one is estimated at circa five mil- lion years (17-19). If this estimate were correct, the ori- gin of L. chinensis should be estimated in the quarter- nary period at least circa 1.25 million years ago, because the mean genetic distance between the populations in Okinawa and Ishigakijima and other area is 0.2492. Livistona chinensis does not appear to have flourished in Aoshima since the warm tertiary period. Though it ap- pears to have originated in Aoshima during the ice age in the quarternary period, the development of L. chinen- sis was not apparently unduly retarded. It is likely that L. chinensis originally possesses cold resistance; it is known to have grown in Tsushima island (latitude 34 degrees north), which is located to the south of the Korean Peninsula as well, suggesting that it thrived through the ice age.

ACKNOWLEDGMENT

This work was supported by a Grant-in-Aid for Scientific Research from the President of Miyazaki University, Dr. Koji Hutagami.

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