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Systematics and Phytogeography |
3Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo 112-0001, Japan; 4Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan
Received for publication March 25, 2005. Accepted for publication December 6, 2005.
ABSTRACT
The present study documents cpDNA variation in the Aristolochia kaempferi group (Aristolochiaceae), which consists of one Chinese and all Japanese and Taiwanese species of the subgenus Siphisia. In a phylogenetic analysis based on the nucleotide sequences of the matK gene, and the atpB-rbcL and trnS-trnG intergenic spacer regions, 38 haplotypes were recognized in the A. kaempferi group and as many as 24 within A. kaempferi. This is the most haplotypes reported for a single species to date. Although six highly significant major clades were identified in the phylogenetic analysis, they were not congruent with previous classifications. This might be attributed to the specific speciation process, such as convergent evolution, incomplete lineage sorting, and/or reticulate evolution. The six major clades had a clear geographical distribution pattern and were significantly associated with geographical distribution of haplotypes in a nested clade analysis and AMOVA. The results allow us to deduce a scenario in which multiple contractions and expansions of the geographical ranges brought about by Quaternary climatic oscillations affected the patterns of genetic diversity. The present geographic patterns of haplotype distribution within the A. kaempferi group can be explained by the last postglacial range expansion from different refugia, and the boundaries may be suture zones.
Key Words: Aristolochiaceae • atpB-rbcL • climatic oscillation • Japan and Taiwan • matK • nested clade analysis • phylogeography • trnS-trnG
The family Aristolochiaceae is a member of the order Piperales, one of the earliest diverging lineages of the angiosperms (Qiu et al., 1999
; Soltis et al., 2000
; Borsch et al., 2003
). It has been divided into two subfamilies: Asaroideae, which includes Asarum and Saruma, and Aristolochioideae, which includes the lineages Aristolochia sensu lato and Thottea sensu lato (Kelly and González, 2003
). In its broad sense, Aristolochia L. consists of about 400 species containing twining lianas, shrubs, and tuberous herbs, that are distributed from tropical to temperate areas worldwide. This genus has frequently been recognized as a distinct genus consisting of the subgenera Aristolochia, Siphisia, and Pararistolochia (Schmidt, 1935
; Ma, 1989
; González, 1999
; González and Stevenson, 2000
). González and Stevenson (2002)
recently divided Aristolochia sensu lato into four genera within two subtribes: Aristolochia sensu stricto and Pararistolochia in the Aristolochiinae, and Endodeca and Isotrema in the Isotrematinae. Under these guidelines, two species of the subgenus Siphisia from southern North America were divided into Endodeca, while the rest of the species of the subgenus Siphisia were contained within Isotrema. However, many species within the subgenus Siphisia still do not have status in Isotrema. To avoid nomenclatural confusion, we used the genus Aristolochia and the subgenus Siphisia in this study.
Phylogenetic relationships in the genus Aristolochia have been studied at various taxonomic levels (Murata et al., 2001
; Sugawara et al., 2001
; González and Stevenson, 2002
; Kelly and González, 2003
; Neinhuis et al., 2005
). Using nucleotide sequences of the matK gene of chloroplast DNA, Murata et al. (2001)
performed the most comprehensive phylogenetic study on the genus Aristolochia to date, although the molecular phylogenetic position of Pararistolochia was determined more recently by Neinhuis et al. (2005)
. Murata et al. (2001)
reported a well-resolved phylogenetic tree and clearly showed two major lineages in Aristolochia that corresponded to the subgenera Siphisia and Aristolochia. In the subgenus Siphisia clade, a well-supported monophyletic group consisting of all Japanese and Taiwanese species and one Chinese species was found. These species included A. cucurbitifolia Hayata, A. kaempferi Willd., A. liukiuensis Hatusima, A. mollissima Hance, A. onoei Franch. et Savat. ex Koidz., and A. shimadai Hayata. We refer to this monophyletic group as the "Aristolochia kaempferi group" in this study.
Some species included in the A. kaempferi group were confused in their species circumscription. Because delicate floral part characters are difficult to observe in dried herbarium specimens, the leaf shape has been used as a major taxonomic character to distinguish between species. However, a wide range of variation in this character has frequently been suggested (Hwang, 1981
; Hamada, 1989
; Kigawa, 2001
; Shiraiwa, 2003
). This has caused taxonomic confusion between A. kaempferi and A. onoei, A. shimadai and A. onoei, and A. shimadai and A. liukiuensis (Liu and Lai, 1976
; Hwang, 1981
, 1988
; Satake and Momiyama, 1982
; Hamada, 1989
; Hou, 1996
; Kigawa, 2001
; Shiraiwa, 2003
). Murata (2006)
recently revised the classification of these species for Japanese flora based mostly on floral characters (Fig. 1). The most significant alteration arising from the study was to merge A. onoei and A. shimadai under the name A. shimadai. Part of A. liukiuensis from the southern islands of Ryukyu was also recognized as A. shimadai. According to his report, A. shimadai has a wide distribution range from Taiwan to Ryukyu, with disjunctive populations in northern Kyushu and central Honshu. In the present study, we basically employed the classification systems of Murata (2006)
. As such, the A. kaempferi group now includes the following five species and one variety of the subgenus Siphisia: A. kaempferi var. kaempferi is a Japanese endemic found in Honshu, Shikoku, and Kyushu; A. kaempferi var. tanzawana S. Kigawa is a Japanese endemic found only in some mountain areas of Honshu; A. liukiuensis is endemic to the northern part of the Nansei Islands (the Amami, Tokunoshima, and Okinawa Islands); A. shimadai is distributed in central Honshu, Kyushu, the southern part of the Nansei Islands (the Ryukyu Islands) in Japan and in Taiwan; A. cucurbitifolia is a Taiwanese endemic found in southern Taiwan; and A. mollissima is a Chinese endemic (taxonomic key in Fig. 1, distribution map in Fig. 2).
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) supported the monophyly of the A. kaempferi group well, phylogenetic relationships within the species were not well resolved. In the clade of the A. kaempferi group, a single sample of A. kaempferi and a clade of other species with branch lengths of less than one step (bootstrap value 70%) were sisters to each other. The latter clade consisted of six terminal branches of zero or only one step. Along with A. liukiuensis, A. onoei, and A. shimadai, morphologically quite distinct species of A. cucurbitifolia (from Taiwan) and A. mollissima (from China) were included in the latter clade. In A. liukiuensis, A. onoei (= A. shimadai in Murata [2006]
and this study), and A. shimadai, from which three, five, and four samples of different origins were used, respectively, more than two haplotypes were recognized, while one haplotype was shared by all three species and A. mollissima. This suggested inconsistencies between current classifications on a morphological basis and the matK phylogeny.
The distribution range of the A. kaempferi group in Japan and Taiwan is almost consistent with the areas of warm-temperate forests. These areas include southern coastal regions of the Japanese main islands that were considered refugia of warm temperate plant species during the last glacial maximum (Kamei et al., 1981
; Tsukada, 1988
). Range expansion from refugia in widely spread species over the Japanese archipelago is an area of interest in phylogeographic studies (for example, Ohi et al., 2003a
, b; Aoki et al., 2004
). However, the geographic structures of haplotypes are mostly inferred without statistical phylogeographic data, such as a nested clade analysis (NCA) (Templeton et al., 1995
; Templeton, 1998
, 2004
) and an analysis of molecular variance (AMOVA) (Excoffier et al., 1992
). Given the high level of intraspecific genetic variation observed in the A. kaempferi group in the matK phylogeny (Murata et al., 2001
), more haplotypes are expected to be obtained if more molecular data and samples are used. This is especially the case with A. kaempferi, which is widely distributed over the Japanese main islands. If we can obtain enough data from the distribution area of the A. kaempferi group, this study will provide both the first opportunity and an excellent one, to perform statistical phylogeographic analyses on the Japanese archipelago.
In considering the present taxonomic and phylogeographic status of the A. kaempferi group, the aims of this study are to verify inconsistencies between cpDNA phylogeny and species boundaries on a morphological basis, to statistically examine the presence of geographical structures, and to infer the phylogeographical process of haplotype distribution in the A. kaempferi group. To achieve this, we used cpDNA sequences of the atpB-rbcL and trnS-trnG intergenic spacer (IGS) regions, as well as the matK gene for our molecular phylogenetic analyses of samples of the A. kaempferi group taken from their entire geographical range in Japan and Taiwan.
MATERIALS AND METHODS
Plant materials
A total of 136 individuals from 94 populations covering the entire distribution range of the A. kaempferi group in Japan and Taiwan and two samples of A. mollissima from eastern China were used in this study (see Appendix). The samples also included those used by Murata et al. (2001)
. Taxa recognition was based on Murata (2006)
, Hou (1996)
for A. cucurbitifolia, and Hwang (1988)
for A. mollissima (Fig. 1). Voucher specimens are preserved in the herbarium of the University of Tokyo (TI). For identification, flowers were collected from one or more individuals in a population. The shape and color of perianth lobes were observed in the field, and flowers were preserved in 70% ethanol for further observation in the laboratory. Aristolochia austroyunnanensis S.M. Hwang, A. kwangsiensis Chun et How ex Liang, A. moupinensis Franch., and A. nakaoi Maekawa, which are closely related to the A. kaempferi group in the matK phylogeny of Aristolochia (Murata et al., 2001
), were used as outgroups.
DNA isolation, polymerase chain reaction (PCR), and sequencing
Leaf fragments collected in the field were dried with silica gel powder and transported to the laboratory. DNA isolation was undertaken as described in Murata et al. (2001)
.
Part of the matK gene and the atpB-rbcL IGS and trnS (GCU)-trnG (UCC) IGS regions of the cpDNA were amplified by PCR using the AF and 8R primers for the matK gene (Ooi et al., 1995
), the atpB and rbcL primers for the atpB-rbcL IGS region (Terachi, 1993
), and the trnS (GCU) and trnG (UCC) primers for the trnS-trnG IGS region (Hamilton, 1999
). The PCR analyses were performed in a 25-µL solution containing 1.0 unit of ExTaq polymerase (TaKaRa Bio, Ohtsu, Shiga, Japan), 2.5 µL of 10% ExTaq buffer (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl and 1.5 mmol/L MgCl2), 2 µL of 0.2 mmol/L dNTP solution, 0.5 µL of 5 pmol/L of each primer, and 10–30 ng of genomic DNA. The PCR cycle consisted of initial denaturation for 1 min at 95°C, followed by 30 cycles of 45 s at 95°C, 1 min at 51°C, 1.5 min at 72°C, and a final extension for 15 min at 72°C.
PCR products were purified using the GeneClean III DNA Purification kit (BIO 101, Carlsbad, California, USA). Purified DNA fragments were used as templates for cycle sequencing with the ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, California, USA) following the manufacturer's instructions. Primers used for PCR of the matK gene and atpB-rbcL IGS and trnS-trnG IGS regions were also used as sequencing primers. Internal primers newly designed for sequencing were 1412F-Ari (5'-ATATAATTCTCATGTATATG-3') and 1470R-Ari (5'-AAGATGTTGATCGTAAACGA-3') for the matK gene and trnS-G580F (5'-AAGGTAAAGCTTTGTTTGA-3') and trnS-G350R (5'-TGTTGYTATTGCTGTTTTCG-3') for the trnS-trnG IGS region. In the cycle sequencing reaction using these primers, the annealing period was changed from 5 to 8 s. DNA sequences were detected using an ABI PRISM 377 DNA sequencer (Applied Biosystems), and all obtained sequence data were deposited in the DNA Data Bank of Japan (DDBJ).
Phylogenetic analyses
All three nucleotide sequences obtained from a sample were manually aligned with others. We employed Simmons and Ochoterena's (2000)
"simple indel coding" for gap coding. Multinucleotide units and multinucleotide repeat units were coded as binary states "0 or 1" in a maximum parsimony (MP) analysis. The gaps caused by the mononucleotide repeat units were removed from consideration in a phylogenetic analysis. This was because homology can be highly uncertain for these repeated nucleotides (Kelchner, 2000
). However, by considering amino acid translations, the mononucleotide repeat units within the matK gene were coded as binary states "0 or 1" (see Appendix S1 Supplemental Data accompanying the online version of this article).
Phylogenetic analyses were performed with PAUP* version 4.0 beta10 (Swofford, 2002
). We conducted an incongruence length difference (ILD) test (Farris et al., 1995
) as implemented in PAUP* for 1000 replicates to test for phylogenetic congruence among the different genes. The MP analysis was conducted using a heuristic search with 100 replicates of random additions, tree-bisection-reconnection (TBR) branch swapping, the MulTrees option, and with no limits for MaxTrees. All characters were equally weighted in the analyses. A strict consensus tree of MP trees was reconstructed, followed by DELTRAN character optimization. A neighbor-joining (NJ) analysis was performed using the evolutionary distance calculated by Kimura's two-parameter method (Kimura, 1980
). For both MP and NJ analyses, bootstrap analyses were performed for 1000 replicates with the same tree search procedure as described.
Nested clade analysis (NCA)
The NCA (Templeton et al., 1995
; Templeton, 1998
, 2004
) was carried out to verify the geographical structure of haplotypes implied within the A. kaempferi group. This is an analytical method for testing the null hypothesis of no haplotype geographical association. The haplotype network was constructed using TCS version 1.18 (Clement et al., 2000
) with 95% confidence limits and the "gaps = missing" option on. Other than for the four outgroup sequences, the same sequence alignment assembled for the MP analysis was used for the network analysis. The gaps coded as binary states "0 or 1" were coded again as binary states "A or T" for use with the TCS. Haplotypes within the inferred networks were nested into hierarchically interlocking groups following the rules provided by Templeton et al. (1987)
and Templeton and Sing (1993)
. A quantitative analysis of geographical data, as described in Templeton (1998)
, was performed using GeoDis 2.2 (Posada et al., 2000
). Historical events underlying the geographical distribution of the A. kaempferi group were inferred using the inference key according to Templeton (2004)
.
Analysis of molecular variance
The AMOVA (Excoffier et al., 1992
) was used to examine the amount of genetic variability partitioned among populations as well as among groups of populations. The analysis was computed based on the matrix of haplotypes data using Kimura's two-parameter distance (Kimura, 1980
). To assess the significance of the various factors that could affect partitioning of genetic variability, populations were grouped according to a set of different grouping criteria: phylogenetic clades, nested clades (nested clade level 3), and species designation. Minimum component of local individuals (mostly one or two individuals) were used as populations. In all analyses, 1000 permutations were run to obtain test statistics using ARLEQUIN 2.000 (Schneider et al., 2000
).
RESULTS
cpDNA sequences
Within the A. kaempferi group, the nucleotide sequence lengths of the three regions were 1169–1175 bp for the matK gene, which corresponded to positions 80–1307 of the complete sequence of the matK gene of Nicotiana tabacum, and 706 bp and 845–916 bp for the atpB-rbcL and trnS-trnG IGS regions, respectively. The gaps coded as binary states "0 or 1" in the MP analysis were one within the matK gene sequence and seven within the trnS-trnG IGS region. No gaps were observed in the atpB-rbcL IGS region within the A. kaempferi group. The number of haplotypes recognized by nucleotide substitutions and coded gaps in the matK gene and the atpB-rbcL and trnS-trnG IGS regions were 22, eight, and 23, respectively. There were no stop codons in any haplotypes of the matK gene. According to the combined sequences obtained from the three regions, 38 haplotypes (36 haplotypes when coded gaps were excluded because haplotypes 10 and 14 were determined only by coded gaps) were recognized for 138 individuals of the A. kaempferi group.
Phylogenetic analyses
The ILD test did not show significant incongruence among pairs of DNA sequence data of the matK gene and the atpB-rbcL and trnS-trnG IGS regions (P value = 1.00 in all three pairs). We performed the following phylogenetic analyses using the combined sequences of the three regions.
Phylogenetic analyses of 38 haplotypes aligned with the four outgroup sequences produced eight MP trees of 132 steps with a consistency index (CI) = 0.902, a retention index (RI) = 0.939, and a rescaled consistency index (RC) = 0.847. The aligned length of the combined sequences of the three regions was 2874 bp including 13 coded gaps and 96 nucleotide substitutions within the A. kaempferi group and the four outgroups (eight coded gaps and 56 nucleotide substitutions within the A. kaempferi group). 57 variable characters were phylogenetically informative. In the strict consensus tree (Fig. 3), which was constructed from the eight MP trees, two major basal sister clades (A and B) with a high bootstrap value were recognized. Clade B was further divided into five well-supported subclades (BI, BII, BIII, BIV, and BV) (Fig. 3). Clade A composed three haplotypes (types 1–3). Clade B composed 35 haplotypes, which formed five subclades (BI–V) that consisted of six (types 4–9), nine (types 10–18), five (types 19–23), one (type 24), and 14 (types 25–38) haplotypes, respectively (Fig. 3). Subclade BV was further divided into three lower level clades, BVa, BVb, and haplotype 38. A NJ tree was reconstructed using the nucleotide sequences of 2702 bp excluding all coded gaps from the combined sequences of the three regions. Because the topology of the NJ tree was almost consistent with the strict consensus tree of the MP trees, the resultant tree is not shown here. Bootstrap values obtained by NJ analysis are shown in parentheses on each branch of the MP trees (Fig. 3).
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Geographical distribution of haplotypes
The geographical distribution of 38 haplotypes was plotted, and the major clades (A–BV) were circled on a map (Fig. 5). These clades appeared to be geographically segregated: haplotypes belonging to clade A were found in the southern Kanto district; subclade BI was disjunctively found between Mt. Tsukuba in the northern Kanto and from eastern Shikoku to the Chubu area; subclade BII was found in the southwestern Chugoku, Setouchi area and northern Kyushu; subclade BIII was found in southern Kyushu; subclade BIV was restricted to southwestern Shikoku; and within subclade BV, BVa was restricted to the Nansei Islands, BVb was restricted to Taiwan, and haplotype 38 was found in China.
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: (1) "past fragmentation and/or long distance colonization" within clade 2-3, (2) "contiguous range expansion" within clades 2-6, 2-11, and 4-3, (3) "restricted gene flow with isolation by distance" within clades 3-2 and 5-2, (4) "long distance dispersal colonization possibly coupled with subsequent fragmentation or past fragmentation followed by range expansion" within clades 3-4 and the total cladogram, and (5) "restricted gene flow/dispersal but with some long distance dispersal over intermediate areas not occupied by the species, or past gene flow followed by extinction of intermediate populations" within clade 4-2.
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, p. 808, 8-YES) This inference would be a suitable explanation for the relationship between 3-2 and 3-3 and/or 3-3 and 3-4, but not for the relationship between 3-2 and 3-4. This is because the extinction of an intermediate population is unlikely between eastern and southwestern Shikoku (between 3-2 and 2-7 within 3-4, Fig. 5). Such an inadequate inference might be affected by the disjunctive geographical distribution of the Nansei Islands and Taiwan. When the NCA was applied to the data set excluding populations in the Nansei Islands and Taiwan, the topology of the network changed. However, other than 2-6 and 3-3, the nested clades recognized by the nesting procedure were the same as those in the original analysis (data not shown). Consequently, although significant levels of geographical association and the types of historical events (Table 1, Populations within the Japanese main islands) were unchanged in the six nested clades (1-4, 2-3, 2-11, 3-2, 4-4, and the total cladogram), historical events inferred within nested clades 4-2b (2-7 and 3-2) and 5-2b (4-2b and 4-3) changed to "long distance dispersal colonization possibly coupled with subsequent fragmentation or past fragmentation followed by range expansion," (p. 808, 13-YES) respectively (a detailed list of Dc, Dn, and I-T values including P levels of other nested clades is shown Appendix S2 Supplemental Data accompanying the online version of this article).
Analysis of molecular variance
The AMOVA (Table 2) shows that there is no relationship between species designations of Murata (2006)
and differentiation in cpDNA variation (the among group variance was 27.46% within all populations from Japan and Taiwan, and 4.63% within populations from the Japanese main islands). However, high proportions of among group variance were obtained when populations were grouped in phylogenetic clades and nested clades (82.53% and 81.66% within all populations from Japan and Taiwan, and 88.29% and 85.54% within populations from the Japanese main islands, respectively).
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Aristolochia kaempferi has a large number of haplotypes
According to the matK phylogeny of the genus Aristolochia by Murata et al. (2001)
, the A. kaempferi group varies considerably. However, because they had low levels of resolution in the phylogenetic tree and fewer samples, they could not make further assumptions regarding this group. Using 2.5 times more nucleotide sequences and 10 times more samples as the previous study (Murata et al., 2001
), we were able to discriminate 38 haplotypes by 56 nucleotide substitutions and eight coded gaps to obtain a well-resolved phylogenetic tree for the A. kaempferi group. It is notable that 24 haplotypes were found for A. kaempferi, which has the widest distribution area in the group, whereas only a single sample was used in the previous study. Although the number of individuals and the amount of molecular data varied in previous studies of Japanese species (Inamura et al., 2000
; Fujii et al., 2002
; Ohi et al., 2003a
, b; Aoki et al., 2004
), the number of haplotypes we found in A. kaempferi is one of the largest found so far for a single species in Japan.
Inconsistencies between morphological grouping and cpDNA phylogeny within the A. kaempferi group
One of the objectives of the present study was to verify inconsistencies between species classification on a morphological basis and cpDNA phylogeny. According to the phylogenetic tree of cpDNA haplotypes obtained in this study, the species classification of Murata (2006)
, which was based mostly on floral characters (Fig. 1), was not consistent with phylogenetic relationships (Fig. 3). The results of the AMOVA (Table 2) also suggested that the current species designations were not compatible with the differentiation at the cpDNA variation. After a comparison with previous taxonomic circumscription of the species, the present cpDNA phylogeny was not found to be consistent with any other classification system of the A. kaempferi group reported to date (Makino and Nemoto, 1925
; Nemoto, 1936
; Makino, 1940
; Hara, 1959
; Ohwi, 1965
; Liu and Lai, 1976
; Okuyama, 1977
; Satake and Momiyama, 1982
; Hwang, 1988
; Hou, 1996
). These inconsistencies may be attributed to the following speciation processes of the A. kaempferi group: (1) convergent evolution of morphological characters, (2) incomplete lineage sorting, and/or (3) reticulate evolution (including hybridization and introgression) within the group. Our preliminary artificial crossing experiments between different morphological species, as well as between different phylogenetic clades, produced seeds in all possible combinations (unpublished data). This at least suggests that pre-zygotic isolation mechanisms are not developed either among taxonomic species or phylogenetic clades in the A. kaempferi group. To clarify the process that produces morphological and molecular variation in the A. kaempferi group, it would be necessary to focus more on nuclear gene genealogy or gene flow at the population level.
Distinct geographical segregation of clades in the Japanese archipelago
The cpDNA phylogeny of the A. kaempferi group reflected geographical distribution rather than taxonomic boundaries (Fig. 5). The six major clades recognized in our phylogenetic tree appear to have a distinct geographical segregation in the visual overlay of haplotypes on the map (Fig. 5). The presence of geographical structure in cpDNA haplotypes has also been reported in recent phylogeographic studies of temperate forest plants on the Japanese main islands (Inamura et al., 2000
; Fujii et al., 2002
; Okaura and Harada, 2002
; Ohi et al., 2003a
, b; Aoki et al., 2004
; Kanno et al., 2004
). However, statistical analyses were rarely employed in these phylogeographic studies. In the present study, we employed NCA and AMOVA to test for the presence of geographical structure and to infer the historical events for observed geographical structure. In the results of the NCA (Table 1) and AMOVA (Table 2), nested clades recognized at nested clade level 3, which are mostly compatible with the six major phylogenetic clades, had statistically significant associations with geographical distribution.
These results supported the further use of NCA to infer historical events for some nested clades. Inference of historical events for some nested clades within the A. kaempferi group included "long distance dispersal and/or colonization" as an alternative historical event. Although the possibility of long-distance dispersal and colonization over long periods cannot be completely eliminated for any plant, it is unlikely for the A. kaempferi group because all species within the group lack characteristic facilities such as elaiosomes or wings. Therefore, we did not consider inferences of the NCA "long distance dispersal and/or colonization" in this discussion.
Using all data sets except for the data from A. mollissima, "past gene flow followed by extinction of intermediate population" was inferred for nested clade 4-2, which included populations from the Japanese main islands, the Nansei Islands, and Taiwan (Table 1, All populations from Japan and Taiwan). This result would be a suitable explanation for the islands' population history because the Nansei Islands were originally connected by a land bridge with Taiwan and Kyushu. Geological data suggest that the connection between the Japanese main islands and the Nansei Islands ceased 1.3–1.7 million years ago (Kimura, 2002
). In due course, the gene flow of the A. kaempferi group between the Nansei Islands and the Japanese main islands might have closed by the early Pleistocene. In the NCA, a higher nesting clade has to be old or older than all the lower level clades nested within it. Hence, the historical events, which were inferred to be higher than nested clade level 3, might also have occurred before the early Pleistocene.
When the NCA was applied to the data set excluding populations in the Nansei Islands and Taiwan, "past fragmentation and range expansion" was inferred for the following nested clade levels: 2-3, 4-2b (2-7 and 3-2), 5-2b (4-2b, and 4-3), and for the total cladogram (Table 1, Populations within the Japanese main islands). This indicates that past fragmentations of populations occurred at different times. Such phylogeographic structures of the A. kaempferi group on the Japanese main islands are considered to reflect multiple contractions/expansions of geographical ranges through Quaternary climatic oscillations, suggested by Hewitt (2000
, 2001
) as being common phylogeographic events in temperate plants in the northern hemisphere.
The geographical patterns of cpDNA haplotypes reported for eight widespread Japanese plants (Aucuba japonica: Ohi et al., 2003a
; Stachyurus praecox: Ohi et al., 2003b
; Fagus crenata: Fujii et al., 2002
; Okaura and Harada, 2002
; Polygonum cuspidatum: Inamura et al., 2000
; Prunus zippeliana, Alpinia japonica, and Elaeocarpus sylvestris var. ellipticus: Aoki et al., 2004
; and the present study) had a small degree of congruence. One common phylogeographical pattern appears amongst Aucuba japonica, Fagus crenata, Quercus spp., and the Aristolochia kaempferi group analyzed in the present study. Although statistical tests were not performed in the former three plants, distinct haplotype groups were commonly found in the Kanto and adjacent areas. In the present study, nested clade 5-1 (equivalent to clade A) is found in the southern Kanto area (Fig. 5). It is evident from fossil pollen data that on the Japanese archipelago during the last glacial maximum (15 000–25 000 years ago), most warmer temperate broadleaf forests migrated to and survived in refugia on the Pacific Ocean side from Kyushu to the Kanto district (Kamei et al., 1981
; Tsukada, 1988
). Taking the common occurrence of distinct haplotype groups in the Kanto and adjacent areas, the existence of one of the major refugia of Japanese temperate flora during the glacial period is suggested in neighboring areas of the Kanto region. The "range expansion" inferred within nested clades 4-2b, 4-3, 5-2b, and the total cladogram, can also be explained by postglacial range expansion from different refugia for the A. kaempferi group. The geographical boundaries among the nested clades may be "suture zones" where different populations meet after a postglacial expansion (Taberlet, 1998
).
APPENDIX
Population and voucher information for this study on the Aristolochia kaempferi group. Voucher specimens are deposited in the herbarium of the University of Tokyo (TI). Note that (cultivated) = cultivated in Medicinal Plant Garden, Setsunan University.
Population number; Taxon; GenBank accessions: matK, atpB-rbcL, trnS-trnG; Location/Source, Coordinates (N/E); Haplotypes; Voucher specimens.
1; A. kaempferi Willd. var. kaempferi; AB180157, AB180105, AB180122; Japan, Kanto, Chiba, 35°10', 140°09'; Type 1; Kana Watanabe, K345, K417.
2; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Kanto, Tokyo, 35°38', 139°14'; Type 1; Kana Watanabe, K290.
3; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Kanto, Kanagawa, 35°19', 139°32'; Type 1; Kana Watanabe, K159.
4; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Kanto, Kanagawa, 35°18', 139°36'; Type 1; Kana Watanabe, K188.
5; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Kanto, Kanagawa, 35°23', 139°10'; Type 1; Kana Watanabe, K100.
6; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Chubu, Shizuoka, 35°15', 138°50'; Type 1; Kana Watanabe, K77.
7; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Chubu, Shizuoka, 35°11', 138°50'; Type 1; Kana Watanabe, K81, K85, Jin Murata, SETS12.
8; A. kaempferi Willd. var.kaempferi; AB180157, AB180105, AB180122; Japan, Chubu, Shizuoka, 35°07', 138°56'; Type 1; Kana Watanabe, K339.
9; A. kaempferi Willd. var.kaempferi; AB180156, AB180105, AB180122; Japan, Chubu, Shizuoka, 35°07', 139°02'; Type 2; Kana Watanabe, K1, K2.
10; A. kaempferi Willd. var.kaempferi; AB180158, AB180105, AB180122; Japan, Chubu, Shizuoka, 34°50', 138°56'; Type 3; Kana Watanabe, K418.
11; A. kaempferi Willd. var.kaempferi; AB180161, AB180108, AB180125; Japan, Chubu, Shizuoka, 35°10', 138°37'; Type 5; Kana Watanabe, K337.
12; A. kaempferi Willd. var.kaempferi; AB180161, AB180108, AB180125; Japan, Chubu, Shizuoka, 34°58', 138°20'; Type 5; Kana Watanabe, K89, K90.
13; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Kinki, Mie, 34°27', 136°47'; Type 7; Kana Watanabe, K408, K412.
14; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Kinki, Wakayama, 33°40', 135°53'; Type 7; Kana Watanabe, K127, K128, K133, K137.
15; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Kinki, Wakayama, 33°48', 135°32'; Type 7; Kana Watanabe, K138, K157.
16; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Kinki, Hyogo, 34°17', 134°52'; Type 7; Kana Watanabe, K364.
17; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Shikoku, Tokushima, 33°54', 134°17'; Type 7; Kana Watanabe, K374.
18; A. kaempferi Willd. var.kaempferi; AB180163, AB180108, AB180128; Japan, Shikoku, Kochi, 33°39', 134°05'; Type 7; Kana Watanabe, K383.
19; A. kaempferi Willd. var.kaempferi; AB180163, AB180110, AB180128; Japan, Kinki, Nara, 34°45', 135°42'; Type 8; Kana Watanabe, K403, K404.
20; A. kaempferi Willd. var.kaempferi; AB180165, AB180108, AB180128; Japan, Shikoku, Tokushima, 33°46', 134°09'; Type 9; Kana Watanabe, K370.
21; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180130; Japan, Chugoku, Hiroshima, 34°21', 132°17'; Type 10; Kana Watanabe, K401.
22; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180130; Japan, Chugoku, Hiroshima, 34°17', 132°17'; Type 10; Kana Watanabe, K420.
23; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180130; Japan, Chugoku, Yamaguchi, 33°53', 132°20'; Type 10; Kana Watanabe, K331.
24; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180130; Japan, Chugoku, Yamaguchi, 34°05', 131°58'; Type 10; Kana Watanabe, K325.
25; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180130; Japan, Kyushu, Ooita, 33°11', 131°47'; Type 10; Kana Watanabe, K311.
26; A. kaempferi Willd. var.kaempferi; AB180166, AB180111, AB180131; Japan, Shikoku, Ehime, 33°58', 133°03'; Type 11; Kana Watanabe, K335.
27; A. kaempferi Willd. var.kaempferi; AB180168, AB180111, AB180130; Japan, Shikoku, Kagawa, 34°31', 134°18'; Type 12; Kana Watanabe, K392
28; A. kaempferi Willd. var.kaempferi; AB180168, AB180111, AB180130; Japan, Chugoku, Okayama, 34°46', 134°06'; Type 12; Kana Watanabe, K399.
29; A. kaempferi Willd. var.kaempferi; AB180167, AB180111, AB180130; Japan, Kyushu, Fukuoka, 33°27', 130°21'; Type 12; Kana Watanabe, K303.
30; A. kaempferi Willd. var.kaempferi; AB180167, AB180111, AB180130; Japan, Kyushu, Saga, 33°25', 130°09'; Type 12; Kana Watanabe, K432.
31; A. kaempferi Willd. var.kaempferi; AB180167, AB180111, AB180130; Japan, Kyushu, Nagasaki, 32°38', 128°46'; Type 12; Kana Watanabe, K448.
32; A. kaempferi Willd. var.kaempferi; AB189731, AB180111, AB180130; Japan, Kyushu, Saga, 33°28', 130°16'; Type 13; Kana Watanabe, K443.
33; A. kaempferi Willd. var.kaempferi; AB180167, AB180111, AB189729; Japan, Kyushu, Nagasaki, 33°12', 129°42'; Type 14; Kana Watanabe, K440.
34; A. kaempferi Willd. var.kaempferi; AB180167, AB180111, AB189729; Japan, Kyushu, Nagasaki, 32°59', 130°04'; Type 14; Kana Watanabe, K444.
35; A. kaempferi Willd. var.kaempferi; AB180168, AB180111, AB180132; Japan, Kyushu, Fukuoka, 33°11', 130°52'; Type 15; Kana Watanabe, K305.
36; A. kaempferi Willd. var.kaempferi; AB180170, AB180111, AB180133; Japan, Kyushu, Kumamoto, 32°58', 130°31'; Type 16; Kana Watanabe, K72, K76.
37; A. kaempferi Willd. var.kaempferi; AB180169, AB180111, AB180133; Japan, Kyushu, Kumamoto, 32°58', 130°32'; Type 17; Kana Watanabe, K70.
38; A. kaempferi Willd. var.kaempferi; AB180171, AB180112, AB180134; Japan, Shikoku, Ehime, 32°56', 132°29'; Type 18; Kana Watanabe, K380.
39; A. kaempferi Willd. var.kaempferi; AB180173, AB180111, AB180135; Japan, Kyushu, Miyazaki, 31°47', 131°17'; Type 19; Kana Watanabe, K55.
40; A. kaempferi Willd. var.kaempferi; AB180172, AB180111, AB180135; Japan, Kyushu, Kagoshima, 31°53', 130°49'; Type 20; Kana Watanabe, K40, K43.
41; A. kaempferi Willd. var.kaempferi; AB180172, AB180111, AB180135; Japan, Kyushu, Kagoshima, 31°52', 130°50'; Type 20; Kana Watanabe, K44.
42; A. kaempferi Willd. var.kaempferi; AB180173, AB180111, AB180136; Japan, Kyushu, Miyazaki, 32°17', 131°27'; Type 21; Kana Watanabe, K50, K54.
43; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180137; Japan, Kyushu, Kagoshima, 31°11', 130°48'; Type 22; Kana Watanabe, K301.
44; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180137; Japan, Kyushu, Kagoshima, 31°31', 130°26'; Type 22; Kana Watanabe, Isaku3.
45; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180137; Japan, Kyushu, Kagoshima, 31°35', 130°33'; Type 22; Kana Watanabe, K66.
46; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180137; Japan, Kyushu, Kagoshima, 31°11', 130°31'; Type 22; Kana Watanabe, K58.
47; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180137; Japan, Kyushu, Kagoshima, 31°11', 130°38'; Type 22; Kana Watanabe, K59, K62, K65.
48; A. kaempferi Willd. var.kaempferi; AB180174, AB180111, AB180135; Japan, Kyushu, Kagoshima, 31°17', 130°24'; Type 23; Kana Watanabe, K179, K180.
49; A. kaempferi Willd. var.kaempferi; AB180175, AB180111, AB180138; Japan, Shikoku, Kochi, 32°44', 133°01'; Type 24; Kana Watanabe, K158, K178.
50; A. kaempferi Willd. var.kaempferi; AB180175, AB180111, AB180138; Japan, Shikoku, Kochi, 33°28', 133°03'; Type 24; Kana Watanabe, K381.
51; A. kaempferi Willd. var.kaempferi; AB180175, AB180111, AB180138; Japan, Shikoku, Ehime, 33°03', 132°40'; Type 24; Kana Watanabe, K377.
52; A. kaempferi var. tanzawana S. Kigawa; AB180159, AB180106, AB180123; Japan, Kanto, Yamanashi, 35°36', 139°04'; Type 1; Kana Watanabe, K116.
53; A. kaempferi var. tanzawana S. Kigawa; AB180159, AB180106, AB180123; Japan, Kanto, Kanagawa, 35°35', 139°11'; Type 1; Kana Watanabe, K120.
54; A. kaempferi var. tanzawana S. Kigawa; AB180159, AB180106, AB180123; Japan, Kanto, Kanagawa, 35°25', 139°08'; Type 1; Kana Watanabe, K92, K93, K96, Jin Murata, A2734.
55; A. kaempferi var. tanzawana S. Kigawa; AB180159, AB180106, AB180123; Japan, Kanto, Kanagawa, 35°32', 139°08'; Type 1; Kana Watanabe, K320.
56; A. kaempferi var. tanzawana S. Kigawa; AB180160, AB180107, AB180124; Japan, Kanto, Ibaraki, 36°13', 140°05'; Type 4; Kana Watanabe, K102, K104, K256, K257.
57; A. kaempferi var. tanzawana S. Kigawa; AB180162, AB180107, AB180126; Japan, Chubu, Shizuoka, 34°59', 137°52'; Type 6; Kana Watanabe, K165.
58; A. kaempferi var. tanzawana S. Kigawa; AB180160, AB180107, AB180127; Japan, Chubu, Shizuoka, 34°51', 137°35'; Type 7; Kana Watanabe, K169.
59; A. kaempferi var. tanzawana S. Kigawa; AB180160, AB180107, AB180127; Japan, Chubu, Aichi, 35°11', 137°08'; Type 7; Kana Watanabe, K297.
60; A. shimadai Hayata; AB180164, AB180109, AB180129; Japan, Kinki, Hyogo, 34°47', 135°14'; Type 7; Kana Watanabe, K360.
61; A. shimadai Hayata; AB180164, AB180109, AB180129; Japan, Kinki, Hyogo, 34°46', 135°14'; Type 7; Kana Watanabe, Maki1, Setu2, Jin Murata, SETS7.
62; A. shimadai Hayata; AB189730, AB189727, AB189728; Japan, Kyushu, Nagasaki, 33°13', 129°48'; Type 14; Kana Watanabe, K437.
63; A. shimadai Hayata; AB180177, AB180114, AB180140; Japan, Nansei-islands, Okinawa, 26°21', 126°43'; Type 25; Kana Watanabe, K3.
64; A. shimadai Hayata; AB180177, AB180114, AB180140; Japan, Nansei-islands, Okinawa, 26°22', 126°46'; Type 25; Kana Watanabe, K6, K39.
65; A. shimadai Hayata; AB180178, AB180114, AB180143; Japan, Nansei-islands, Okinawa, 26°22', 126°45'; Type 28; Kana Watanabe, K8.
66; A. shimadai Hayata; AB180177, AB180114, AB180146; Japan, Nansei-islands, Okinawa, 24°34', 124°18'; Type 29; Kana Watanabe, K278, K280.
67; A. shimadai Hayata; AB180177, AB180114, AB180146; Japan, Nansei-islands, Okinawa, 24°22', 124°07'; Type 29; Kana Watanabe, K285, K286.
68; A. shimadai Hayata; AB180177, AB180114, AB180146; Japan, Nansei-islands, Okinawa, 24°23', 123°44'; Type 29; Kana Watanabe, K264, K267.
69; A. shimadai Hayata; AB180177, AB180114, AB180146; Japan, Nansei-islands, Okinawa, 24°23', 123°43'; Type 29; Kana Watanabe, K268, Jin Murata, SETS41.
70; A. shimadai Hayata; AB180177, AB180114, AB180148; Taiwan, Taipei, 25°09', 121°30'; Type 30; Kana Watanabe, AS2, TW005.
71; A. shimadai Hayata; AB180177, AB180114, AB180148; Taiwan, Changhua, 23°50', 120°37'; Type 30; Kana Watanabe, B20, B75–1, B75–2.
72; A. shimadai Hayata; AB180181, AB180114, AB180148; Taiwan, Taichung, 24°15', 121°15'; Type 33; Kana Watanabe, AS3.
73; A. shimadai Hayata; AB060760, AB180114, AB180148; Taiwan, Nantou, 24°05', 121°10'; Type 33; Jin Murata, SETS38.
74; A. shimadai Hayata; AB060763, AB180117, AB180147; Taiwan, Pingtung, 21°55', 120°50'; Type 31; Kana Watanabe, C-1 64, F32, Jin Murata, SETS65.
75; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Kagoshima, 28°21', 129°29'; Type 25; Kana Watanabe, AMA, Jin Murata, SETS43, SETS44.
76; A. liukiuensis Hatusima; AB060757, AB180113, AB180139; Japan, Nansei-islands, Kagoshima, 28°07', 129°15'; Type 25; Jin Murata, SETS18.
77; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 27°41', 128°55'; Type 25; Kana Watanabe, K126.
78; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 27°02', 127°57'; Type 25; Kana Watanabe, K12, K16.
79; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 26°45', 128°16'; Type 25; Kana Watanabe, K28.
80; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 26°41', 127°59'; Type 25; Kana Watanabe, K11.
81; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 26°38', 127°54'; Type 25; Kana Watanabe, K25, Jin Murata., SETS34.
82; A. liukiuensis Hatusima; AB180176, AB180113, AB180139; Japan, Nansei-islands, Okinawa, 26°12', 127°15'; Type 25; Kana Watanabe, K235.
83; A. liukiuensis Hatusima; AB180176, AB180113, AB180142; Japan, Nansei-islands, Okinawa, 26°45', 128°19'; Type 27; Kana Watanabe, OKN.
84; A. liukiuensis Hatusima; AB180176, AB180113, AB180141; Japan, Nansei-islands, Okinawa, 26°36', 128°08'; Type 26; Kana Watanabe, K27.
85; A. liukiuensis Hatusima; AB180179, AB180113, AB180144; Japan, Nansei-islands, Okinawa, 26°10', 127°49'; Type 28; Kana Watanabe, K251.
86; A. liukiuensis Hatusima; AB060746, AB180113, AB180144; Japan, Nansei-islands, Okinawa, 26°05', 127°43'; Type 28; Jin Murata, SETS40.
87; A. liukiuensis Hatusima; AB180176, AB180113, AB180145; Japan, Nansei-islands, Okinawa, 26°22', 127°59'; Type 29; Kana Watanabe, K243, K244.
88; A. liukiuensis Hatusima; AB180176, AB180113, AB180145; Japan, Nansei-islands, Okinawa, 26°23', 127°59'; Type 29; Kana Watanabe, Isaku4.
89; A. cucurbitifolia Hayata; AB060763, AB180115, AB180147; Taiwan, Pingtung, 22°12', 120°51'; Type 32; Kana Watanabe, K455.
90; A. cucurbitifolia Hayata; AB180183, AB180115, AB180150; Taiwan, Chiayi, 23°30', 120°40'; Type 34; Kana Watanabe, AC2.
91; A. cucurbitifolia Hayata; AB060741, AB180115, AB180149; Taiwan, Kaohsiung, 22°58', 120°41'; Type 35; Kana Watanabe, B65, Jin Murata, SETS39.
92; A. cucurbitifolia Hayata; AB060741, AB180115, AB180149; Taiwan, Chiayi, 23°33', 120°43'; Type 35; Kana Watanabe, AC3.
93; A. cucurbitifolia Hayata; AB201052, AB180115, AB180148; Taiwan, Chiayi, 23°33', 120°39'; Type 37; Kana Watanabe, K457.
94; A. cucurbitifolia Hayata; AB060741, AB201053, AB180148; Taiwan, Taitung, 23°04', 121°08'; Type 36; Kana Watanabe, K461.
95; A. mollissima Hance; AB180184, AB180116, AB180151; China (cultivated); Type 38; Kana Watanabe, AM2, Jin Murata, SETS30. A. austroyunnanensis S. M. Hwang; AB060740, AB180120, AB180153; China, Guangxi (cultivated); Outgroup; Jin Murata, SETS15. A. kwangsiensis Chun & How ex Liang; AB060744, AB180121, AB180154; China, Guangxi (cultivated); Outgroup; Jin Murata, SETS28. A. moupinensis Franch.; AB060751, AB180118, AB180152; China, Yunnan (cultivated); Outgroup; Jin Murata, SETS9. A. nakaoi Maekawa; AB060752, AB180119, AB180155; Nepal (cultivated); Outgroup; Jin Murata, SETS14.
FOOTNOTES
1 The authors thank Dr. T. Ohi-Toma for his technical advice and helpful comments, Prof. C.-F. Hsieh and Mr. T. Siraiwa for their support in our fieldwork, and Dr. H. Murata and Dr. T. Sugawara for providing samples. The authors acknowledge funding for this project from the Sasakawa Scientific Research Grant from The Japan Science Society (15-253 and 16-287K) to K.W., and a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture (No. 12440240) to J.M. 
2 Author for correspondence (e-mail: kana{at}bg.s.u-tokyo.ac.jp
)
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