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(American Journal of Botany. 2003;90:1645-1652.)
© 2003 Botanical Society of America, Inc.

Systematics and Phytogeography

Distinct geographic structure as evidenced by chloroplast DNA haplotypes and ploidy level in Japanese Aucuba (Aucubaceae)1

Tetsuo Ohi2, Tadashi Kajita and Jin Murata

Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo 112-0001, Japan

Received for publication July 11, 2002. Accepted for publication April 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The geographic distribution of diploid and tetraploid cytotypes and cpDNA haplotypes throughout the entire range of Aucuba japonica was investigated. We measured relative genome size using flow cytometry and sequenced two cpDNA intergenic regions, atpB-rbcL and psbA-trnH (GUG). Two haplotypes include both diploids and tetraploids; four others are all tetraploids. Based on the combination of these haplotypes and cytotypes, eight "haplo-cytotypes" can be defined, which show a distinct geographic structure. Two diploid haplo-cytotypes are distributed in the southwestern part and six tetraploid ones in the northeastern part of the Japanese archipelago. Diploid and tetraploid haplo-cytotypes with the same haplotype are, in one case, disjunctively distributed, and in another case, in contact. The phylogenetic relationships of haplo-cytotypes indicate that the traditional circumscription of A. japonica is paraphyletic. One lineage consists of four tetraploid haplo-cytotypes and another of diploid and tetraploid haplo-cytotypes plus A. chinensis. Tetraploidization occurred independently at least three times, once at the base of the former lineage and twice in the latter. Taking the geographic, cytological, and phylogenetic evidence into account, the formation of the present geographic differentiation pattern of haplo-cytotypes through postglacial expansion from glacial refugia is discussed.

Key Words: atpB-rbcL • Aucuba japonica • Aucubaceae • geographic structure • Japanese archipelago • phylogeography • polyploid • psbA-trnH (GUG) • tetraploidization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The Japanese archipelago consists of four main islands (Hokkaido, Honshu, Shikoku, and Kyushu) and is located at the eastern edge of East Asia (Fig. 1). The archipelago stretches 3000 km from northeast to southwest over a wide range of climatic zones from cool-temperate to subtropical and has a complex mountainous topography along the central axis of Honshu. The Sea of Japan side is characterized by a heavy snowfall in winter and the Pacific Ocean side by a milder and dry climate in winter. A number of Japanese plant taxa have been divided into two floristic groups according to their gross distribution patterns on the Sea of Japan side or the Pacific Ocean side (Kanai, 1958 ; Hotta, 1974 ). The difference in environmental conditions between the two regions has been suggested to have led to adaptive differentiation among closely related species or intraspecific taxa (Fukuoka, 1966 ; Hotta, 1974 ).



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  		Fig. 1. Present distribution of Aucuba japonica (bold line) and names of regions and districts in the Japanese archipelago

 
Aucuba japonica Thunb. (Aucubaceae), an evergreen dioecious shrub, is one of the species with infraspecific taxa that differ between the Sea of Japan side and the Pacific Ocean side (Fukuoka, 1966 ; Hotta, 1974 ). In the geographic range of the genus Aucuba, which extends from the Himalayan region through south China and Taiwan to the Japanese archipelago, A. japonica is distributed at the easternmost edge (Kanai, 1963 ; Hara, 1966 ). This species is common and widely distributed in the temperate forest zone from southwestern Hokkaido to Okinawa Island in the Japanese archipelago (Hara, 1966 , 1989 ; Noshiro, 1999 ) (Fig. 1). The southern and northwestern edges of its geographic distribution extend to the northernmost part of Taiwan and to the Ullundo and Chejudo Islands of Korea, respectively (Li and Yang, 1993 ; Lee, 1996 ). In recent floras of Japan, three varieties—A. japonica var. japonica, A. japonica var. borealis Miyabe et Kudô, and A. japonica var. ovoidea Koidz.—have been recognized based on the combination of morphological differences, ploidy levels, and geographic distribution (Hara, 1989 ; Noshiro, 1999 ). Here, we refer to the infraspecific taxa by their varietal epithet alone. Variety japonica is a tetraploid (2n = 4x = 32) and is generally considered a member of the Pacific Ocean side, distributed from the Tohoku district to the Kii Peninsula of Honshu and eastern Shikoku. Variety borealis is a tetraploid, with decumbent shoots, smaller leaves, and hairy young buds and inflorescences. This variety is considered a member of the Sea of Japan side and is thought to be morphologically adapted to the heavy snowfall region in southwestern Hokkaido and the Sea of Japan side in Honshu (Fukuoka, 1966 ; Hara, 1966 ; Hotta, 1974 ). Variety ovoidea is morphologically quite similar to var. japonica, but it is a diploid (2n = 2x = 16) distributed from the Chugoku district of Honshu, western Shikoku, and Kyushu to Okinawa Island.

As outlined, both Hara (1989) and Noshiro (1999) rather clearly delimited the geographic distribution of three varieties of A. japonica that not only morphologically differentiated between the Pacific Ocean side (var. japonica) and the Sea of Japan side (var. borealis), but also cytologically differentiated between the southwestern part (diploid: var. ovoidea) and the northeastern part (tetraploids: var. japonica and var. borealis) of the Japanese archipelago. However, the geographic boundary between diploid and tetraploid cytotypes is overemphasized as they are based on the cytological studies of Kurosawa (1971 , 1976 , 1979 ) in which the sampling localities were quite limited. More extensive surveys of the geographic distribution of cytotypes are necessary. Moreover, morphologically intermediate forms frequently appear in the middle part of Honshu, especially around the contact zones of the geographic distribution of the varieties (e.g., Kitamura and Murata, 1971 ; Satomi, 1975 ). A survey of genetic markers of A. japonica is expected to provide us with a better understanding of the geographic distribution and evolutionary history of the varieties.

The objective of the present study is to obtain an insight into the geographic structure of A. japonica by phylogeographic analysis. We surveyed ploidy levels using flow cytometry (FCM) and cpDNA haplotypes using nucleotide sequence data from two intergenic spacer (IGS) regions throughout the whole geographic range of A. japonica. Specific questions of the study were (1) Is the geographic boundary between diploid and tetraploid cytotypes clearly delimited? (2) Are the geographic boundaries between diploid and tetraploid, if any, and between the Sea of Japan side and the Pacific Ocean side also recognized by the spread of cpDNA haplotypes? and (3) What is the evolutionary history of the geographic distribution pattern suggested by the cpDNA haplotype phylogeny?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant materials
The sampling strategy was to collect a few individuals from 124 localities that sufficiently covered the whole geographic range of A. japonica. In total, 199 individuals were collected (see Appendix, available as Supplementary Data accompanying the online version of this article). Of these, 120 individuals were collected as cuttings and planted in the greenhouse at the Botanical Garden, Graduate School of Science, University of Tokyo, for use in both sequence analysis and determination of the ploidy level. The remaining 79 individuals were collected as silica-gel-dried material with voucher specimens and only used for sequencing analysis. Four other species from the genus, A. chinensis Benth., A. himalaica Hook. f. et Thoms., A. eriobotryifolia Wang, and A. omeiensis Fang, from China and Taiwan were also used for phylogenetic analysis (Table 1). Garrya ovata Benth. (Garryaceae), which is closely allied to Aucuba according to the molecular phylogeny of angiosperms of Soltis et al. (2000) , was used as an outgroup (Table 1).


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  		Table 1. Sample information for other Aucuba species and an outgroup species from outside Japan

 
Determination of ploidy levels
Chromosome numbers of a few individuals were preliminarily counted by regular methods. Fresh root tips were pretreated with 0.01% colchicine solution at 4°C for 5–6 h and fixed with 45% acetic acid solution for 10–15 min. After being macerated in 30% HCl at 60°C for 1 min, root tips were stained with 1% acetic orcein solution for overnight. They were squashed and observed under a light microscope, and chromosome numbers were counted. A diploid and a tetraploid (BG: 83-216 and OT: A003 in the Appendix, available as Supplementary Data accompanying the online version of this article) with clear chromosome numbers in the count were used as external standards for the following analysis.

To examine ploidy levels of all individuals, we used FCM. A fresh fragment of mature leaf (about 2 x 2 cm) was chopped with a razor blade in 320 µL chopping buffer (10 mmol/L Tris-HCl [pH 8.0], 2 mmol/L MgCl2, 1.0% TritonX-100, 8 µL mercaptoethanol, 32 mg PVP [polyvinylpyrrolidone], and 3.2 µL RNase) on a plastic petri dish, and a further 250 µL chopping buffer was added. Each step was performed on ice. The buffer, containing isolated nuclei, was filtered through a CellTrics 30 µm mesh filter (Pertec, Münster, Germany) and then stored at 37°C for 10 min. The isolated nuclei were stained with 50 µL propidium iodide solution for a few hours. The flow cytometer was a FACscan, working with Cellquest software (Becton Dickinson, Franklin Lakes, New Jersey, USA). We initially ran the diploid standard and set the peak position of the fluorescence histogram at 130 (relative value) by adjusting the instrument's detector and amps setting (ampGain of FSC, 3.00; voltage of SSC, 320; ampGain and voltage of FL2, ~2.00 and ~365, respectively). At these settings, the peak position of the fluorescence histogram of the tetraploid standard was 258. At the beginning of each course of experiments, which consisted of analyzing 20–30 individuals, we ran the diploid standard and slightly adjusted its peak position on the fluorescence histogram to 130. Ploidy level of each individual was interpreted by comparing the peak position of the fluorescence histogram with those of the external diploid and tetraploid standards.

DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing
Prior to genomic DNA extraction, leaf tissue ground in liquid nitrogen was washed using the HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) method (Setoguchi and Ohba, 1995 ). Genomic DNA was extracted from a washed leaf pellet, using the CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle, 1987 ) with a slight modification. After preliminary screening using five IGS regions (T. Ohi and J. Murata, unpublished data), two IGS regions were utilized as markers in the present analysis: (1) atpB-rbcL, with primers (forward: 5'-AAGTAGTAGGATTGGTTCTCTA-3') and (reverse: 5'-TAGTTTCTGTTTGTGGTGACAT-3') (Terachi, 1993 ), and (2) psbA-trnH (GUG), with primers (forward: 5'-CGAAGCTCCATCTACAAATGG-3') and (reverse: 5'-ACTGCCTTGATCCACTTGGC-3') (Hamilton, 1999 ). The PCR reaction mixture consisted of 1.0 unit of ExTaq polymerase (TaKaRa Bio, Ohtsu, Shiga, Japan), 3 µL of 10% ExTaq buffer (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, 1.5 mmol/L MgCl2), 2.4 µL of 0.2 mmol/L dNTP solution, 1.0 µL of 10 pmol/L of each primer, and 10–30 ng of genomic DNA, to a total volume of 30 µL. The PCR cycle conditions were 96°C (1 min); then 33 cycles of 96°C (1 min), 53°C (1 min), 72°C (1 min); and finally 72°C (10 min). The PCR products were purified with the GeneClean III DNA purification Kit (BIO 101, Carlsbad, California, USA), following the instructions provided by the manufacturer. Purified template DNA fragments were amplified using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA) with the same primers as those used for PCR. DNA sequencing was performed using an ABI PRISM 377 DNA sequencer (Applied Biosystems). Sequence data was deposited in the DNA Data Bank of Japan (DDBJ).

Phylogenetic analysis
Multiple sequence alignment was performed manually. The criteria of alignment and mutational interpretations were made with reference to the criteria of Golenberg et al. (1993) and Kelchner (2000) . Based on the combination of cpDNA haplotypes and ploidy levels, "haplo-cytotypes" were determined and used as operational taxonomic units (OTUs) in the following analysis. To evaluate the diversity of the analyzed IGS regions, the nucleotide diversity ({pi}; Nei, 1987 ) among haplotypes in each region was calculated based on the Jukes and Cantor model (Jukes and Cantor, 1969 ) using DnaSP version 3.53 (Rozas and Rozas, 1999 ). Length polymorphisms were treated as missing data. A maximum-parsimony analysis was conducted using PAUP* version 4.0 beta10 (Swofford, 2002 ) and the branch and bound search option. The length polymorphisms were used as a fifth character and scored as binary states "0 or 1," but length polymorphisms next to the polynucleotide tracts (polyA or polyT) were excluded from the analysis. All characters were treated as unordered and equally weighted. Branch support was assessed by bootstrap analysis with 1000 replicates using the branch and bound search option. Genetic distances, using substitution data only, were calculated based on the Jukes and Cantor model and a neighbor-joining (NJ) tree was reconstructed using PAUP*. The branch support for the NJ tree was also assessed by bootstrap analysis with 1000 replicates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Ploidy levels
Chromosome numbers of the two individuals used as external standards for FCM were confirmed with light microscopy (Fig. 2, I). Their fluorescence histograms are shown in Fig. 2, II. The peak positions of the fluorescence histograms obtained from all individuals analyzed by FCM fit clearly into one of the two states. The relative fluorescence value of each state were 133.15 ± 6.77 and 253.79 ± 11.80 (mean ± SD). The latter value was about twice the former, and the values for the diploid and tetraploid standards were in the range of each state, respectively. All individuals were therefore interpreted as diploid (2x) or tetraploid (4x). Of 120 individuals, 46 individuals (38.3%) were interpreted as diploid and 74 (61.7%) as tetraploid (see Appendix, available as Supplementary Data accompanying the online version of this article).



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  		Fig. 2. Somatic metaphase chromosomes of two individuals of Aucuba japonica used as external standards (I), and fluorescence histograms of their relative genome size stained by propidium iodide using a FACscan flow cytometer (II). (a) BG: 83-216, diploid (2n = 2x = 16), (b) OT: A003, tetraploid (2n = 4x = 32). Detailed data for individuals (peak positions/profiles) are given in the Appendix, available as Supplementary Data accompanying the online version of this article

 
CpDNA sequences of intergenic spacer regions
All the sequence data obtained in the present study were deposited in DDBJ under accession numbers AB087780–AB087788 (atpB-rbcL sequences) and AB087789–AB087800 (psbA-trnH sequences). Within A. japonica, lengths of nucleotide sequences of the two regions were 727 base pairs (bp) for atpB-rbcL and 403–413 bp for psbA-trnH, in which four and five nucleotide substitutions were included, respectively (Tables 2, 3). There were no length polymorphisms in atpB-rbcL, while one length polymorphism, consisting of a 10-bp tandem repeat formed by a duplication of neighbor sequences, was present in psbA-trnH (Tables 2, 3). Among accessions including other species of Aucuba, the length of nucleotide sequences of the two regions varied from 726 to 727 bp for atpB-rbcL and from 379 to 415 bp for psbA-trnH, with 13 and 14 nucleotide substitutions, respectively. Four length polymorphisms in the range of 10–24 bp were observed in psbA-trnH (Tables 2, 3), two of which were duplications of neighboring sequences (length polymorphism footnotes "c" and "d" in Table 2) and others were deletions. The values of nucleotide diversity ({pi}) of the two regions were 0.0032 in atpB-rbcL and 0.0065 in psbA-trnH within A. japonica and 0.0054 in atpB-rbcL and 0.0125 in psbA-trnH within the genus Aucuba (Table 3).


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  		Table 2. Variable nucleotide sites of atpB-rbcL and psbA-trnH sequences in the genus Aucuba. Sequences are numbered from the 5' end to the 3' end in each region. Dots (.) indicate that the character states are the same as for A. japonica haplotype Ja. In psbA-trnH, "1a" to "1d" indicate length polymorphisms, with "0" indicating absence and "1" presence; "—" in position 333 indicates an unsequenced part because of the length polymorphism of position 318–341 in haplotype H2

 

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  		Table 3. The characterization of sequences of two intergenic regions, atpB-rbcL and psbA-trnH, within Aucuba japonica and the genus Aucuba

 
According to the combined sequences obtained from the two IGS regions, six haplotypes were recognized for 199 individuals of A. japonica (Table 2). The number of individuals for each haplotype were 46 (23.1%; Ja), 67 (33.7%; Ov), 75 (37.7%; B1), 2 (1.0%; B2), 3 (1.5%; B3), 6 (3.0%; B4). In addition, intraspecific haplotype variation was also observed in A. chinensis in Taiwan (C1 and C2) and A. himalaica (H1 and H2) (Table 2).

Phylogenetic relationships of haplo-cytotypes
Diploid and tetraploid individuals were found in both the Ja and Ov haplotypes. All individuals with either B1, B2, B3, or B4 haplotype were tetraploids. Based on the combination of cpDNA haplotypes (Ja, Ov, B1, B2, B3, B4) and ploidy levels (diploid [2x], or tetraploid [4x]), eight haplo-cytotypes (Ja-2x, Ja-4x, Ov-2x, Ov-4x, B1-4x, B2-4x, B3-4x, B4-4x) were determined for 120 individuals (see Appendix, available as Supplementary Data accompanying the online version of this article).

The aligned length of the combined sequences of atpB-rbcL and psbA-trnH was 1173 bp. Of 54 variable characters, including length polymorphisms, 20 were phylogenetically informative. A single most parsimonious (MP) tree of 55 steps with consistency index (CI) = 0.965, retention index (RI) = 0.962, and rescaled consistency index (RC) = 0.929 was obtained (Fig. 3). The haplo-cytotypes of A. japonica did not form a monophyletic group, but formed a clade (bootstrap probability [BP] 82%) that included two haplotypes (C1 and C2) of A. chinensis. This clade was comprised of two subclades. The first consisted of haplo-cytotypes Ja-2x, Ja-4x, Ov-2x, Ov-4x, and the two haplotypes of A. chinensis (BP 72%; the JOC lineage). The second subclade consisted of haplo-cytotypes B1-4x, B2-4x, B3-4x, and B4-4x (BP 92%; the B lineage). A neighbor-joining (NJ) tree was reconstructed using the sequences of 1069 bp, excluding length polymorphisms. As the topology of the tree was identical with that of the MP tree, the resultant tree is not shown here. Bootstrap probabilities for the NJ tree are shown in parenthesis on each branch of the MP tree (Fig. 3).



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  		Fig. 3. The single most parsimonious tree based on the sequences of atpB-rbcL and psbA-trnH (length = 55 steps, consistency index = 0.965, retention index = 0.962, rescaled consistency index = 0.929) from the genus Aucuba. Bootstrap probabilities (BP) and branch lengths indicated above branches and below branches. The BP of the neighbor-joining method also is indicated in parenthesis above each branches. Arrows indicate tetraploidization events

 
We calculated the approximate dates of earliest divergence events between the JOC and B lineages, within the JOC lineage, and within the B lineage from nucleotide sequences data, using the general nucleotide substitutions rate of cpDNA and IGS regions (4.87 x 10–10 to 8.24 x 10–9 substitutions · site–1 · yr–1; Richardson et al., 2001 ). The values of the nucleotide diversity of the combination of atpB-rbcL and psbA-trnH were 0.0037 within the clade of the JOC and B lineages, 0.0013 within the JOC lineage, and 0.0009 within the B lineage. Based on these values, the dates of diversification were ca. 3.80–0.22 million years ago (mya) between the JOC lineage and the B lineage, ca. 1.33–0.08 mya at the base of the JOC lineage, and ca. 0.92–0.05 mya at the base of the B lineage.

Geographic distribution of haplo-cytotypes
The geographic distribution of individuals analyzed was plotted on a map (Fig. 4). All diploid haplo-cytotypes (Ja-2x, Ov-2x) are found in the southwest region of the Japanese archipelago and all tetraploid haplo-cytotypes (Ja-4x, Ov-4x, B1-4x, B2-4x, B3-4x, B4-4x) in the northeast. The geographic ranges of the diploid and tetraploid cytotypes have a distinct boundary, which extends longitudinally between the east side of Oki Island and the middle of Shikoku (ca. 134° E). The haplo-cytotype B1-4x occupies a considerably larger area than the other tetraploid haplo-cytotypes (Ja-4x, Ov-4x, B2-4x, B3-4x and B4-4x). Four tetraploid haplo-cytotypes (Ov-4x, B2-4x, B3-4x and B4-4x) are relatively restricted to particular localities. Amongst the diploids, the Ov-2x haplo-cytotype occupies a wider geographic area than the Ja-2x haplo-cytotype.



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  		Fig. 4. The geographic distribution of eight haplo-cytotypes of Aucuba japonica in Japan, Taiwan, and Korea based on the combination of cpDNA haplotypes and ploidy levels (Ja-2x, Ja-4x, Ov-2x, Ov-4x, B1-4x, B2-4x, B3-4x, and B4-4x; large symbols). The haplo-cytotypes of individuals for which ploidy levels were not examined were inferred from comparison with haplo-cytotypes in the same population or those of nearby populations and are indicated by small symbols. The two haplotypes of A. chinensis in Taiwan (C1 and C2) are indicated by two kinds of star symbols. Thin lines are the boundaries of the geographic distribution of major haplo-cytotypes, and the thick line is the geographic boundary between diploid and tetraploid cytotypes

 
The B1-4x haplo-cytotype appears in the Tohoku and Hokuriku districts, the Sea of Japan side of the Chugoku district, and on the Pacific Ocean side in the Kii Peninsula and eastern Shikoku. Three rare haplo-cytotypes (B2-4x, B3-4x, B4-4x), which are in the B lineage, occur in restricted localities within the range of B1-4x; B2-4x in the southwest part, B3-4x in the middle part, and B4-4x in the middle and north parts. The Ja-2x and Ja-4x haplo-cytotypes, which have the same cpDNA haplotype but different ploidy levels, are disjunctively distributed between a part of Kyushu and the Kanto and Chubu districts. The Ov-2x haplo-cytotype is distributed not only in parts of Kyushu, Shikoku, and the Chugoku district, but also in the Nansei Islands, Ullundo Island of Korea, and the northernmost part of Taiwan. The Ov-4x haplo-cytotype appears only at the eastern edge of the range of Ov-2x in Shikoku and the Chugoku district.

The 79 individuals, ploidy levels of which were not examined, were also plotted on the map, with inference of their ploidy level made by comparison with haplo-cytotypes in the same population or those of nearby populations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Geographic distribution of cytotypes
The analysis of ploidy level throughout the whole geographic range of A. japonica shows that a distinct geographic boundary separates the diploid and tetraploid cytotypes (Fig. 4). This boundary extends longitudinally, close to 134° E. The position of the boundary is similar to that suggested by Kurosawa (1971 , 1976 , 1979 ), but until now it has not been confirmed.

In many plant species, populations with different ploidy levels are geographically segregated and often parapatric (e.g., Stebbins, 1971 ; Lewis, 1980 ). The geographic segregation of diploid and polyploid cytotypes has sometimes been explained by their adaptive selection to environments. Polyploids have been suggested to be better colonizers and more adaptive to new habitats than their ancestral diploids. This capacity may then further promote the geographic segregation of diploid and polyploid (Stebbins, 1971 ; Lewis, 1980 ; Thompson and Lumaret, 1992 ; Soltis and Soltis, 2000 ). For Japanese Aucuba, however, no clear-cut topographic or climatic difference now exists to fit the geographic boundary between diploid and tetraploid cytotypes (Japan Meteorological Agency, 1993 ). Thus, the segregation of cytotypes of the species may originate from nonadaptive factors such as postglacial expansion of plants from different glacial refugia (Thompson and Lumaret, 1992 ; Van Dijk et al., 1992 ; Van Dijk and Bakx-Schotman, 1997 ). This possibility in the evolution of Japanese Aucuba is discussed later.

Divergence of intergenic spacer regions
Noncoding regions of cpDNA, i.e., IGS and intron regions, are suitable for the analysis of evolutionary processes at lower taxonomic levels (Soltis and Soltis, 1998 ). It is often more effective to utilize a combination of several selected noncoding regions to increase the number of characters available for analysis, because the evolutionary rate may differ among regions, and some regions lack phylogenetic information (Sang et al., 1997 ; Small et al., 1998 ; Kim et al., 1999 ). As a consequence of the preliminary screening of five IGS regions, two IGS regions, atpB-rbcL and psbA-trnH, were selected for use in the present study. The nucleotide diversity ({pi}) of psbA-trnH was greater than that of atpB-rbcL both within the species and within the genus Aucuba. The nucleotide diversity within A. japonica (0.0032 in atpB-rbcL; 0.0065 in psbA-trnH) is lower than those within Cunninghamia lanceolata (0.0074 in trnD-trnT), C. konishii (0.0118 in trnD-trnT) (Lu et al., 2001 ), Kandelia candel (0.0265 in trnL-trnF) (Chiang et al., 2001 ), and Michelia formosana (0.0106 in atpB-rbcL) (Lu et al., 2002 ) and is higher than or similar to those seen within Fagopyrum statice (0.0022 in trnK 5' intron, and 0.0049 in trnK 3' intron) and F. leptopodum (0.0032 in trnK 5' intron, and 0.0014 in trnK 3' intron) (Ohsako and Ohnishi, 2001 ). The nucleotide diversity of A. japonica, therefore, appears to be typical for the intraspecific level.

Haplo-cytotypes and taxonomic implications
The geographic boundary between diploid and tetraploid cytotypes did not correspond with the spread of cpDNA haplotypes because representatives of both the Ja and Ov haplotypes are found on either side of the geographic boundary of cytotypes. Moreover, the boundary between the Sea of Japan side and the Pacific Ocean side is also not recognized by the spread of cpDNA haplotypes alone. The B1-4x haplo-cytotype, which was mainly distributed in the Sea of Japan side, appears on the Kii Peninsula and eastern Shikoku (Fig. 4). Nevertheless, the geographic distribution of haplo-cytotypes, determined by combined haplotypes and ploidy levels, does show a distinct geographic structure across the Japanese archipelago (Fig. 4).

In the most recent taxonomic treatment of A. japonica, three varieties have been defined based on the combination of morphological differences, ploidy level, and geographic distribution (Hara, 1989 ; Noshiro, 1999 ). The distinct geographic boundary between the diploid and tetraploid cytotypes coincides with the apparent geographic boundary between var. ovoidea (diploid) and the two other tetraploid varieties (var. borealis and var. japonica). Moreover, the geographic ranges of haplo-cytotypes B1-4x and Ja-4x obtained in the present study are generally consistent with the presumed geographic ranges of var. borealis and var. japonica, which are thought to represent the Sea of Japan side element and the Pacific Ocean side element, respectively (Kanai, 1958 ; Fukuoka, 1966 ; Hotta, 1974 ).

However, there are a number of interesting exceptions to the general agreement between the traditional circumscription of varieties and the geographic distribution of haplo-cytotypes defined in the present study. Plants on the Kii Peninsula and eastern Shikoku that have been treated as var. japonica have the B1-4x haplo-cytotype, which is the major haplo-cytotype on the Sea of Japan side, allying them with var. borealis. Plants in Kyushu that have been treated as var. ovoidea (diploid) include haplo-cytotype Ja-2x as well as Ov-2x, but the Ja-2x has the same cpDNA haplotype as plants in the geographically distant Kanto and Chubu districts (Ja-4x), which have been treated as var. japonica. The geographic boundary between haplo-cytoypes of the B lineage and Ov-4x lies parallel to and east of the geographic boundary of diploid and tetraploid cytotypes (Fig. 4).

Phylogenetic relationships of haplo-cytotypes and origins of tetraploids
The phylogenetic relationships of haplo-cytotypes based on the sequences of atpB-rbcL and psbA-trnH indicate that A. japonica is paraphyletic because A. chinensis of Taiwan was nested in the JOC lineage (Fig. 3). In the recent flora of Taiwan, A. chinensis is clearly distinguished from A. japonica by narrower leaves with large, coarse teeth on the margin as well as by the length of inflorescence and pedicel (Li and Yang, 1993 ). Our results suggest the possibility of rapid morphological divergence in A. chinensis or of chloroplast capture between A. japonica and A. chinensis via hybridization.

The B lineage consists solely of tetraploid haplo-cytotypes (B1-4x, B2-4x, B3-4x, B4-4x) in the phylogenetic tree, while the JOC lineage consists of haplotypes Ja and Ov, which include both diploid and tetraploid cytotypes (Ja-2x and Ja-4x; Ov-2x and Ov-4x), plus the diploid A. chinensis with chromosome number 2n = 2x = 16 (Hara, 1966 ; Kurosawa, 1971 ) (Fig. 3). Other Aucuba species, i.e., A. himalaica, A. omeiensis, and A. eriobotryifolia, are all diploids (Hara, 1966 ; Kurosawa, 1971 , 1981 ) (Table 2). The arrangement of ploidy levels on the phylogenetic tree suggests that tetraploidization occurred independently at least three times, once at the base of the B lineage and once in each of haplotypes Ja and Ov of the JOC lineage (Fig. 3). Because each of the haplo-cytotypes Ja-4x and Ov-4x has the same cpDNA haplotype as their sister diploid (Ja-2x and Ov-2x), these two tetraploidizations might be relatively more recent events than that of the B lineage in which three mutational sites were included (Fig. 3).

Phylogeography within Aucuba japonica
Many plant phylogeographic studies using cpDNA have detected a distribution of intraspecific haplotypes that is geographically structured (e.g., Soltis et al., 1997 ; Comes and Kadereit, 1998 ; Taberlet et al., 1998 ; Hewitt, 1999 , 2000 ; Petit et al., 2002 ). These studies inferred that the geographic structuring was a consequence of a contraction into refugia during the last glacial period (approximately 100 000–10 000 yr ago), followed by a postglacial expansion.

During the last glacial period, although the Japanese archipelago was not covered by a major ice sheet (Ono, 1984 ), the mean annual temperature was 5°–9°C cooler and the precipitation was less than at present (Yasuda and Narita, 1981 ; Tsukada, 1988 ). In addition, as a result of the lower sea levels (ca. 100 m below present), Shikoku and Kyushu were continuous with Honshu, and the continental shelf, ca. 20–30 km from the present coastline, emerged around the archipelago (Ohta and Yonekura, 1987 ). Reconstructions of past Japanese vegetation based on palynological data suggested that the archipelago during the last glacial maximum (25 000–15 000 yr ago) was almost entirely covered by coniferous forests (Abies, Picea, Pinus, and Tsuga). Most of the warmer temperate broadleaf forests migrated to and survived in restricted areas (refugia) on the Pacific Ocean side from Kyushu to the Kanto district (Kamei et al., 1981 ; Tsukada, 1988 ). As the climate warmed, they recovered and expanded northward or to higher altitudes (Tsukada, 1988 ; Takahara et al., 2000 ).

As with plant species of warmer temperate broadleaf forests, A. japonica's range should have contracted into such refugia scattered on the coast of the Pacific Ocean side during the glacial period. The present geographic distribution pattern of cpDNA haplotypes within A. japonica could be explained as the result of expansion from refugia after the last glacial period, which can partly be traced by the emergence and increase of the pollen of Aucuba in postglacial sediments in non-refugial areas (Tsuji and Suzuki, 1977 ; Nakabori, 1981 ; Tsukada, 1988 ; Inada et al., 1998 ). Taking into account the geographic, cytological, and phylogenetic evidences obtained in the present study, the formation of the present geographic distribution of haplo-cytotypes through postglacial expansion from glacial refugia is hypothesized as follows.

The tetraploidization in the B lineage is probably the earliest of the three tetraploidizations within Japanese Aucuba, occurring at least before the last glacial maximum, as we calculated ca. 0.92–0.05 mya for the earliest divergence within the lineage in the previous section. For the B lineage, the B1-4x haplo-cytotype is the most widespread in the archipelago at present (Fig. 4). It may be that this tetraploid haplo-cytotype has rapidly expanded from refugia on the Pacific Ocean side into the Sea of Japan side through the middle part of Honshu, where the mountains were not high enough to prevent the expansion. This process might be promoted by adaptation of the plant with the B1-4x haplo-cytotypes to a heavy snowfall in the Sea of Japan side.

The Ja-2x and Ja-4x haplo-cytotypes share the same haplotype, but are disjunctively distributed in Kyushu and the Kanto and Chubu districts (Fig. 4). This distribution pattern may be the result of postglacial expansions from different refugia in each area.

The geographic boundary between B1-4x and Ja-4x haplo-cytotypes coincides with the topographical features of the Japanese archipelago, i.e., the mountain ranges (altitude 2000–3000 m) surrounding the Kanto and Chubu districts in central Honshu. The mountain ranges probably acted as a barrier to the northward expansion by the Ja-4x haplo-cytotype from the Kanto and Chubu districts or to southward migration by the B lineage from the Sea of Japan side.

The geographic ranges of Ov-2x and Ov-4x haplo-cytotypes are in contact, and their geographic boundary is also the geographic boundary of cytotypes (Fig. 4). The Ov-4x haplo-cytotype is restricted to a small area at the eastern edge of its sister diploid. This proximity suggested a more recent origin of Ov-4x than that of Ja-4x, which is disjunct from its putative progenitor. The Ov-4x haplo-cytotype might have arisen at the leading edge of expansion of Ov-2x from refugia in Kyushu and/or Shikoku where it remains today.

Further studies are necessary to confirm in detail the process and evolutionary history of tetraploidization in A. japonica.

Conclusion
The results of the present study, which surveyed ploidy levels and cpDNA haplotypes throughout the whole geographic range of A. japonica, show a clear geographic differentiation between diploids in the southwestern part and tetraploids in the northeastern part of the archipelago, but neither strictly supports the differentiation of A. japonica between the Pacific Ocean side and the Sea of Japan side in the traditional phytogeographic view, nor the traditional intraspecific circumscription. The geographic structure and the phylogenetic relationship of haplo-cytotypes suggest a complicated evolutionary history for Japanese Aucuba, involving at least three independent tetraploidizations and postglacial expansion from separate refugia by the several haplo-cytotypes. Such research on other Japanese plant taxa will provide new insights into the phytogeography of the Japanese archipelago.


   FOOTNOTES
 
1

 The authors thank Prof. A. Toe for use of a FACScan, Prof. C.-F. Hsieh and Prof. Y. Tateishi for their support of our field work, Dr. M. Mishima for technical advice, Prof. H. Ohashi, Dr. H. Murata, Dr. T. Kurosawa, Dr. Y. Iokawa, Dr. J.-H. Park, and Dr. H. Kudo for collecting, and Mr. G. Kenicer for reading the manuscript. The present study was partly supported by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture (No. 09041165 and 13375003 to J. Murata). Back

2 E-mail: ooi{at}bg.s.u-tokyo.ac.jp Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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