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(American Journal of Botany. 2008;95:885-896.) doi: 10.3732/ajb.2007331 © 2008 Botanical Society of America, Inc. |
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Systematics and Phytogeography |
Botanical Gardens, Graduate School of Science, University of Tokyo, Tokyo 112-0001, Japan
Received for publication 18 October 2007. Accepted for publication 1 May 2008.
ABSTRACT
Hybridization via distributional changes should be an important factor for plant speciation. Previous cpDNA analyses of the Aristolochia kaempferi group, comprising six taxa in East Asia, showed a distinct phylogeographic structure resulting from distributional changes brought about by paleoclimatic oscillations. However, the cpDNA phylogeny was incongruent with morphologically defined taxa. To explore the evolutionary processes responsible for the inconsistency between cpDNA and morphology, we made artificial crosses and performed phylogenetic analyses using multiple nuclear markers. All crosses among different taxa or cpDNA clades set fruit, if crossing direction is not considered. The five nuclear phylogenies mostly did not support either the taxa or the cpDNA clades. A combined analysis of cpDNA and the PI exon revealed the two major lineages in the group, lacking a prezygotic isolating barrier between them. However, an asymmetric prezygotic isolating barrier occurs between populations of the Japanese main islands and of other areas that belong to different cpDNA subclades. It seems reasonable to conclude that the development of a prezygotic isolating mechanism is not necessarily proportional to the degree of genetic divergence. These results suggested that species boundaries within the group are blurred due to speciational processes associated with multiple hybridization and introgression resulting from repeated contacts among differentiated populations.
Key Words: APETALA3 • Aristolochiaceae • crossing experiments • East Asia • PhyA • PISTILLATA • reticulate evolution • secondary contact
Over the last decade, knowledge of intraspecific phylogeographic patterns of cpDNA variation has been greatly enhanced for numerous plant taxa worldwide (reviewed in Avise, 2000
; Hewitt, 2004). Phylogeographic studies have provided valuable insights into the historical processes that have influenced current population structure. The climatic changes of the Quaternary affected not only plant species’ geographical distribution but also patterns of genetic diversity (Comes and Kadereit, 1998; Hewitt, 2000; Petit et al., 2003). A change in climatic regimes that resulted in widespread glaciation forced organisms into refugia and subsequently led to population fragmentation. During this period, the effects of genetic drift, including population bottlenecks and founder effects, were amplified by highly restricted gene flow, resulting in distinct genetic lineages within populations. Glacial retreat is also thought to have affected rapid population range extension through leading edge expansion and secondary contact between populations derived from different refugia, initiating gene flow.
Phylogeographic relationships among closely related species and species complexes have been proposed via geographically structured cpDNA variations across species in various genera (McKinnon et al., 2001; Petit et al., 2002; Jakob and Blattner, 2006; Frajman and Oxelman, 2007). In many cases, conclusive evidence for the cooccurrence of different species sharing the same cpDNA variation has not been found. Incomplete lineage sorting, convergent evolution, and/or reticulate evolution (including hybridization and introgression) provide one explanation. In some studies, the incongruence between morphological discrimination and cpDNA differentiation has been interpreted in light of hybridization resulting from secondary contact between different species. Paleoclimatic oscillations are proposed to have facilitated these contacts (Hewitt, 2001) and therefore to have played an important role in evolutionary history via hybridization (Mallet, 2007).
The Aristolochia kaempferi group (Aristolochiaceae) is a monophyletic clade in the lineage possessing pipe-like flowers with a three-lobed gynostemium (Murata et al., 2001; Ohi-Toma et al., 2006). This group comprises six deciduous perennial lianas in Japan, Taiwan, and eastern China. Morphological features associated with geographic distributions delimited the taxa. Floral features including perianth size, shape, and color are the primary diagnostic characters distinguishing the four taxa (A. kaempferi var. kaempferi Willd., A. kaempferi var. tanzawana Kigawa, A. liukiuensis Hatusima, A. shimadai Hayata), and the other two taxa (A. cucurbitifolia Hayata, A. mollissima Hance) are clearly distinguished in the group by the presence of specialized leaves (see Fig. 1 in Watanabe et al., 2006). Molecular phylogenetic relationships within the A. kaempferi group were not well resolved.
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analyzed cpDNA variation across the entire geographic range of the group. The results revealed two distinct clades (A and B) and five subclades within clade B, incongruent with recognized taxonomic boundaries. Four taxa in Japan and Taiwan (not including the Chinese species A. mollissima) were polyphyletic and share some cpDNA haplotypes (Fig. 1A, updated from Watanabe et al., 2006). These clades and subclades demonstrated distinct geographic segregation (Fig. 1B), and nested clade phylogeographical analyses (NCPA) suggested that the geographical patterns reflect a series of range contractions and expansions brought about by Quaternary climatic oscillations. This pattern of cpDNA variation, which is geographically structured and not consistent with the current taxonomic classification, might be the result of incomplete lineage sorting, convergent evolution, or hybridization and introgression between species. Because it is difficult to determine the evolutionary processes responsible for this kind of complex diversification based on cpDNA genetic analysis alone, nuclear DNA analysis and crossing experiments should be applied to elucidate the diversity of the A. kaempferi group.
In phylogeography, cpDNA has utility as a molecular marker for investigating population genetics and geographical patterns due to haploidy, uniparental inheritance, and a short coalescence time. However, cpDNA phylogenies frequently generate unresolved species relationships and are incongruent with morphological classifications in several plant taxa (reviewed in Wendel and Doyle, 1998). Due to biparental inheritance and a more rapid evolutionary rate than cpDNA, nuclear DNA has the potential to overcome some of the limitations of the cpDNA. However, there are practical and theoretical limitations in nuclear DNA analysis (Schaal and Olsen, 2000; Hare, 2001). Nuclear ribosomal regions, such as ITS, are commonly used in plant phylogenetic studies, but often divergence levels are low among closely related species and are claimed to be problematic as a result of multicopies and concerted evolution (Álvarez and Wendel, 2003). Although analysis of single- or low-copy nuclear genes is a proposed solution (Sang, 2002; Álvarez and Wendel, 2003; Zhang and Hewitt, 2003), few single or low-copy nuclear genes have been explored for evolutionary studies (Small et al., 2004). Furthermore, the presence of paralogs, needed to identify individual alleles, and sequence recombination are difficulties that need to be overcome (Sang, 2002; Zhang and Hewitt, 2003; Small et al., 2004). Several recent studies have established the utility of multiple low-copy nuclear genes and the value of their combined use with cpDNA markers (e.g., Cronn et al., 2003; Oh and Potter, 2003; Howarth and Baum, 2005; Syring et al., 2005).
While molecular phylogenetic approaches are the most effective means to reconstruct population history and detect gene flow among populations, the existence of reproductive isolating barriers cannot be demonstrated with DNA data. Conducting artificial crossing experiments in plants is generally straightforward (Lowe et al., 2004). Reproductive isolation can be broadly categorized into premating isolation, postmating and prezygotic isolation, and postzygotic isolation (Coyne and Orr, 2004). Artificial crosses, used to detect prezygotic and postzygotic isolation, can also indicate past or recent natural hybridization events (Aguilar et al., 1999). Thus, experimental crosses can support inferences obtained from molecular data.
The current study explores the evolutionary processes in the A. kaempferi group that may be responsible for inconsistency between morphology-based classifications and cpDNA phylogenetic relationships. To assess reproductive isolation and potential hybridization among morphological taxa and cpDNA subclades, we conducted a four-year artificial crossing experiment at the Botanical Gardens, the University of Tokyo (BGUT). To investigate whether a nuclear DNA phylogeny reflected groups defined by morphological characters or geographically segregated cpDNA subclades, we analyzed newly developed genealogies of multiple nuclear genes in the A. kaempferi group. On the basis of these analyses, the evolutionary history of the A. kaempferi group is discussed.
MATERIALS AND METHODS
Plant materials
Two-hundred two individuals from 119 populations representing the geographic distribution of the A. kaempferi group in Japan and Taiwan and two individuals of A. mollissima representing one population in eastern China were used for cpDNA analysis. Of these, 66 individuals were new collections from which genomic DNA was extracted, and the remainder had been used in previous studies (Murata et al., 2001; Watanabe et al., 2006). The following names were applied to the morphological taxa: Kaempferi (A. kaempferi var. kaempferi), Tanzawana (A. kaempferi var. tanzawana), Shimadai (A. shimadai), Liukiuensis (A. liukiuensis), Cucurbitifolia (A. cucurbitifolia), and Mollissima (A. mollissima). Based on Murata et al. (2001) and Ohi-Toma et al. (2006), A. manshuriensis Com., A. moupinensis Franch., A. saccata Wall., and A. westlandii Hemsl., were chosen as outgroups.
Of 204 individuals used for cpDNA analysis, 72 were used in cross-fertility experiments at the BGUT, and 51 of these 72 were successfully transplanted from the field to the BGUT. Flowers of the remaining 19 individuals were collected from natural habitats as pollen sources only. The number of flowers per individual was correlated with plant size. After two years of growth, plants were more than 1 m tall and had one to several flowers. The 51 transplanted individuals had more than two years of growth.
In view of the six cpDNA subclades and six morphological taxa, 16 representative samples were chosen from 204 individuals, and five regions from three nuclear loci were resolved. Sample numbers and other information (in parentheses: population number, taxon, and cpDNA clade) are as follows: K160 (3, Kaempferi, A); K642 (96, Kaempferi, A); K580 (17, Kaempferi, BI); K325 (24, Kaempferi, BII); K61 (47, Kaempferi, BIII); K158 (49, Kaempferi, BIV); K95 (54, Tanzawana, A); K653 (111, Tanzawana, A); K555 (110, Tanzawana, BI); Setu2 (61, Shimadai, BI); K437 (62, Shimadai, BII); K282 (66, Shimadai, BV); AS2 (70, Shimadai, BV); K32 (118, Liukiuensis, BV); AC2 (90, Cucurbitifolia, BV); and AM2 (95, Mollissima, BV). One nuclear DNA region, including an additional 45 samples, which covers the entire distribution range of the A. kaempferi group in Japan and Taiwan, was also extensively analyzed. Voucher information and GenBank accession numbers of all 204 individuals and four outgroups are in Appendix S1 (see Supplemental Data accompanying online version of this article).
cpDNA sequencing and phylogenetic reconstruction
To assess the cpDNA relationships of the 66 newly collected samples to the subclades identified by Watanabe et al. (2006), we sequenced the nucleotides for the matK gene, and the atpB-rbcL and trnS-trnG spacer regions using the procedure of Watanabe et al. (2006). The methods of these authors were also used for aligning the resulting nucleotide sequences and for scoring indels.
Phylogenetic analyses were conducted using PAUP* version 4.0 beta10 (Swofford, 2002), including maximum parsimony (MP) and maximum likelihood (ML; Felsenstein, 1973) methods. MP analysis was performed as reported previously (Watanabe et al., 2006). A strict consensus tree of MP trees was generated, and then character changes were reconstructed on the tree with DELTRAN character optimization using PAUP*. In ML analyses, the best-fit model and parameter values were estimated by the hierarchical likelihood ratio test (Felsenstein, 1988) in Modeltest version 3.06 (Posada and Crandall, 1998). Maximum likelihood heuristic searches were conducted using 100 replicates with random sequence addition. Because of the enormous amount of time required for bootstrap analyses using the ML method, a "Fast" stepwise-addition bootstrap with 1000 replicates was performed in PAUP*. The data matrices and the MP and ML trees are available from the TreeBASE database (study accession number S2057; http://www.TreeBASE.org).
Cross-fertility experiments
Similar to other Aristolochia species, the A. kaempferi group has protogynous flowers. The stigmatic papillae mature the day the flowers open. Two or three days later, the anthers dehisce after the stigma branch has extended and the sticky stigma has dried. The phenology in the field differs among populations because of regional climatic variation. Depending on the number of flower buds, an individual flowers for two or three months because flowers open consecutively. Flowering is earlier toward the south. For example, the peak is March in the Nansei Islands but June in the Kanto region. At BGUT, the controlled greenhouse environment resulted in the same flowering period (April to June 2003–2007) among all transplanted individuals. Pollen remained viable for two weeks when stored at 5°C with moisture control. All crossing experiments were conducted at BGUT.
Self-incompatibility and selfing mechanisms need to be investigated before successful interspecific crosses can be generated. In tests for geitonogamy, 44 flowers of 15 individuals at the female stage were self-pollinated by other flowers of the same individual. Although their flowers are strongly protogynous, if the female stage overlaps with the male stage even a little, autogamy can occur. To evaluate autogamy, 113 flowers from 27 individuals at the female stage were left without hand-pollination.
Diallel crosses were performed among individuals representing the six morphological taxa and six cpDNA subclades. For cross-fertility experiments, mesh bags were used to enclose all flower buds. After the flowers opened, 464 flowers of 53 individuals at female stage, and 
500 flowers of 62 individuals at male stage, were cross-pollinated. When seeds were obtained, fruit sets per number of treatments (F/N) and their percentages were evaluated.
Nuclear DNA markers
Based on low copy number and ease of primer design, the nuclear-encoded phytochrome A (PhyA) gene of the phytochrome gene family, and the B-class genes APETALA3 (AP3) and PISTILLATA (PI) of the MADS-box gene family, were selected to deduce phylogenetic relationships in the A. kaempferi group. The PhyA gene serves a primary role in red/far-red light perception and signal transduction (Nagatani et al., 1993). The AP3 and PI genes are involved in petal and stamen identity, but their homologs in Aristolochia are expressed in the stamen and the perianth tube (Jaramillo and Kramer, 2004). Notably, the AP3 and PI genes are not expressed in the perianth limb, which provides the most important character for distinguishing morphological taxa in the A. kaempferi group. To amplify the PhyA homolog, we used the primers of Ohi-Toma et al. (2006) and other primers designed during this study (Table 1). RNA was required for the exon regions of the AP3 and PI homologs, and genomic DNA was needed for the remaining regions. The characterized sequences were verified for similarity using a BLAST search of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
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. For PCR amplification, the annealing temperature for the PI intron was changed from 51°C to 57°C. PCR products were purified using the GeneClean III DNA purification kit (BIO 101, Carlsbad, California, USA) and DNA sequences detected using ABI PRISM BigDye Terminator version 3.1 and an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, California, USA), following the manufacturer’s instructions. With direct sequencing of several different samples, overlapping double peaks or superimposed peaks were found at the same sites for both complementary strands, revealed by the application of AutoAssembler (Applied Biosystems). To separate these heterogeneous fragments, we performed single-strand conformation polymorphism (SSCP) analyses, using a modification of the method of Ebihara et al. (2005). The original PCR products were electrophoresed (300 V) in 0.5x MDE gel (Cambrex Bio Science, Rockland, Maine, USA) containing 0–10% glycerol at 15–25°C for 15 h (PhyA) or 13 h (AP3 and PI). The acrylamide gel was stained with SYBR Gold nucleic acid gel stain (Invitrogen, Carlsbad, California, USA). Each band was excised with a razor blade and submerged overnight in 20 µL of distilled water (DW). This DW solution was used directly as a template for PCR with 1 pmol/L of each primer, following the same program as for the former reaction. The PCR product was then used as a template for the cycle sequencing reaction and was subsequently sequenced using the method described earlier. The original PCR products, in which the heterogeneous fragments could not be separated by SSCP, were secondarily cloned using a TOPO TA Cloning Kit (Invitrogen). At least 16 clones per sample were picked and sequenced. For the clone sequences, nucleotides that were not detected by direct sequencing were regarded as PCR errors.
Complementary DNA (cDNA) sequencing
RNA was extracted from fresh young flower buds using TRIzol Reagent (Invitrogen). To identify AP3 and PI homologs, we tested a few samples for 3'-rapid amplification of cDNA ends (3'-RACE) with forward primers (Table 1) using the TaKaRa RNA PCR Kit (AMV) version 3.0 (TaKaRa Bio, Ohtsu, Shiga, Japan) and the manufacturer’s instructions. The amplified PCR products were cloned and sequenced using the method described earlier. Based on the results obtained from 3'-RACE, reverse primers were designed for each locus on the 3'-untranslated region (3'-UTR) (Table 1).
For the other representative samples and four outgroups, AP3 and PI were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) as described by Stellari et al. (2004). For an extensive analysis, PI of an additional 45 samples was also evaluated, using SSCP and cloning as described for the genomic DNA analysis.
Tests of recombination
Several methods with different algorithms are appropriate to ascertain the presence of recombination among characterized sequences (Posada and Crandall, 2001; Posada, 2002). In this study, four different methods were chosen to detect recombination. The first method, the informative sites test (Worobey, 2001), was performed in the program PIST version 1.0 (Rambaut and Worobey, 2001). The second method, the partial likelihood assessment through optimization (Grassly and Holmes, 1997), was performed using PLATO version 2.11 (Grassly and Rambaut, 1998). These two methods require a substitution model, parameter values, and a phylogenetic tree with branch lengths. The model and parameter values estimated by the hierarchical likelihood ratio test in Modeltest, the ML topology tree, and branch lengths inferred by PAUP* were contained in the input file for PIST and PLATO. The third method, based on the pairwise homoplasy index statistic (Phi; Bruen et al., 2006), was conducted using the program SplitsTree 4 (Huson and Bryant, 2006). Finally, the recombination analysis using cost optimization was performed with the program Recco version 0.92 (Maydt and Lengauer, 2006).
Genealogical analyses
Alignment of nucleotide sequences including outgroups, and coding of indels followed Watanabe et al. (2006). We conducted an incongruence length difference (ILD) test (Farris et al., 1995) as implemented in PAUP* for 1000 replicates to test for phylogenetic congruence between the exon and intron of a gene. Phylogenetic reconstructions were generated as described in cpDNA analyses. Allele networks were constructed using statistical parsimony (Templeton et al., 1992) and a 95% confidence limit using the program TCS version 1.21 (Clement et al., 2000). In this program, each indel in the matrix was treated as a single mutation event. The data matrices and the MP and ML trees are available from the TreeBASE database (study accession S2057).
RESULTS
cpDNA phylogeny
Thirty-eight cpDNA haplotypes were previously identified from 138 individuals (Watanabe et al., 2006). In this study, an additional 66 individuals were examined, and the former haplotypes were included here along with five new haplotypes. Thus, 43 haplotypes representing 204 individuals identified within the A. kaempferi group. Sequence characteristic details and statistics from the phylogenetic analyses are provided in Table 2. The strict consensus tree of the 24 MP trees (Fig. 1A) was largely consistent with the topology of the ML tree. Two major clades (A and B) and five subclades in clade B (BI–BV) with high bootstrap values were identified. None of the morphological taxa, with the exception of Mollissima, formed monophyletic groups. However, the geographic distribution of the subclades (A, BI–BV) was segregated (Fig. 1B), as demonstrated in a previous study (Watanabe et al., 2006).
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The results of the cross-pollination experiments are summarized in Table 3. The cross-pollination experiments within each morphological taxon resulted in fruit development and seed formation in five morphological taxa (with the exception of Mollissima) (boldfaced text in Table 3A). For cross-pollination experiments between different morphological taxa, all combinations set fruit if crossing direction is not considered. Considering crossing direction, the following combinations resulted in no or low (<15%) fruit set: Kaempferi or Tanzawana (as an ovule parent) with Liukiuensis, Cucurbitifolia, or Mollissima (as a pollen parent); and Shimadai or Cucurbitifolia (as an ovule parent) with Mollissima (as a pollen parent). Moderate fruit set (30.0–66.7%) was observed for the following combinations: Liukiuensis, Cucurbitifolia, or Mollissima (as an ovule parent) with Kaempferi or Tanzawana (as a pollen parent).
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Identification of PhyA, AP3, and PI in representative samples
González and Stevenson (2002) proposed that the genus Aristolochia sensu lato is divided into the subtribe Aristolochiinae and the subtribe Isotrematinae. Their proposed classification was supported by data from molecular phylogenies and chromosome numbers (Neinhuis et al., 2005; Ohi-Toma et al., 2006). For species in the subtribe Aristolochiinae, the PhyA, AP3, and PI genes are single-copy genes, but species in subtribe Isotrematinae have two paralogs for these genes, likely as a result of ancient polyploidization (Mathews and Donoghue, 2000; Jaramillo and Kramer, 2004; Stellari et al., 2004; Ohi-Toma et al., 2006). The A. kaempferi group and outgroups are classified in the subtribe Isotrematinae, suggesting that the PhyA, AP3, and PI genes each included two paralogs in these taxa.
In a preliminary analysis, Kaempferi was found to include two paralogs (paralog 1-1 and paralog 1-2) of the PhyA gene, and paralog 1-2 was isolated using locus-specific primers (Ohi-Toma et al., 2006). In the present study, the locus sequences were determined using the same primers. The cDNA analyses revealed that both the AP3 and PI genes from the A. kaempferi group included two copies of sequences homologous to copies detected in A. manshuriensis (Jaramillo and Kramer, 2004), referred to here as AP3 locus 1 and AP3 locus 2 and PI locus 1 and PI locus 2. Comparison of the cDNA and genomic DNA sequences was used to identify the position of exons and introns of these loci. The locus-specific reverse primers in cDNA designed in this study resolved two loci for each of the AP3 and PI genes (Table 1 and Fig. 2). In the genomic DNA analyses, the differences in fragment length in agarose gel electrophoresis indicated the presence of two loci for each gene. RT-PCR amplifications of AP3 locus 1 and PI locus 1 did not generate any bands in agarose gel electrophoresis for 10 of 16 representative samples analyzed. Because the intron regions detected for these loci were exceedingly variable, the resulting nucleotide sequences were difficult to align. Therefore, both AP3 locus 1 and PI locus 1 were excluded from the A. kaempferi group phylogeny reconstruction (data not shown). Herein, PhyA paralog 1-2, AP3 locus 2, and PI locus 2 will be abbreviated PhyA, AP3, and PI, respectively.
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Recombination tests and nuclear DNA phylogenies
The presence of recombination was evaluated using four different tests before inferring the nuclear DNA phylogeny of each of five regions from 16 representative samples (Table 4). No evidence for recombination was detected in the exon of PhyA, but at least one test indicated recombination events for the other four regions. Both the Phi and PLATO tests indicated recombination in the PI exon. Recombination in the exon/intron of AP3 and in the intron of PI was indicated by the PLATO and Phi tests, respectively. Because the recombination tests were equivocal, analyses of all data sets were conducted assuming no recombination.
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The statistics obtained from the phylogenetic analyses of the five nuclear regions are summarized in Table 2. Percentages of variable and parsimony-informative characters of each region were similar to, or higher than, those of the combined cpDNA values. The topologies of the strict consensus MP and ML phylogenetic trees for each of the five regions were generally identical and had low resolution. The statistical parsimony networks also did not clarify genealogical relationships among the alleles in each region (Appendix S2, see Supplemental Data with online version of this article). Here, only the MP tree for each region is shown (Fig. 3). On the basis of the five nuclear DNA phylogenies, perhaps the morphological taxonomic boundaries were not supported, although the trees were lacking resolution. No clades or subclades detected in the cpDNA phylogeny appeared to be monophyletic in the five nuclear DNA phylogenies, except for subclade BIV within the PhyA tree. Sharing of alleles among different morphological taxa or cpDNA subclades was also observed in the five trees. For example, in the PI exon tree, seven samples (K653, K555, K437, K61, K158, AC2, and AM2) shared an identical allele. Meanwhile, the five nuclear DNA phylogenies had some commonalities. In every tree, there seems to be a distinct "Kaempferi with cpDNA clade A" branch (K160 and K642), which did not form a phylogenetic group with other samples possessing a clade A of cpDNA type (K95 and K653). The "Tanzawana with clade A" samples (K95 and K653) shared alleles with a "Tanzawana with subclade BI" sample (K555) in four trees but not for the AP3 exon.
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In the statistical parsimony network, the 35 haplotypes of alleles in the A. kaempferi group were separated into two groups (
and β lineages) with four outgroups located at intermediate positions between the lineages (Fig. 4). The lineage was composed of nine haplotypes detected in 22 alleles from 13 individuals, and the β lineage was composed of 26 haplotypes in 100 alleles resolved in 52 individuals. Four individuals shared both alleles from the two lineages. In each lineage, the haplotypes detected from the alleles were classified as common or rare based on their quantity (see the color coding in Fig. 4). Two common (red) and seven rare (yellow) haplotypes were identified in the lineage, and two common (blue) and 24 rare (pale blue) haplotypes were identified in the β lineage. The lineage included two morphological taxa (Kaempferi and Shimadai) and the β lineage included all six morphological taxa. The two common haplotypes in the lineage were only observed in Kaempferi, and the two common haplotypes in the β lineage were shared by four and five morphological taxa, respectively (Fig. 4).
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lineage, the common haplotypes were confined to the southern Kanto area, whereas the rare haplotypes were scattered throughout the western part of the Japanese main islands. Both the common and rare haplotypes in the β lineage were widely distributed over the range of the A. kaempferi group, but the area of common haplotypes of the lineage was excluded. The geographic distributions of the common haplotypes of the and β lineages largely corresponded to that of clades A and B from the cpDNA phylogeny, respectively. However, the geographic distributions of some haplotypes of the and β lineages were not entirely consistent with those of clades A and B. All six individuals that had the rare haplotypes of the lineage possessed the haplotypes of clade B, while four individuals that had the haplotypes of the β lineage had the haplotypes of clade A. Furthermore, these six individuals with the rare haplotypes of the lineage were scattered over the geographic range of clade B, and the four individuals of the β lineage were limited to populations ascribed to the cpDNA clades geographic boundary.
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Lack of prezygotic isolating barrier
A previous study addressed the incongruence between morphological taxa and cpDNA phylogenetic subclades and raised the question of whether the taxa or the clades represented the evolutionary history of the A. kaempferi group or whether neither reflected diversification processes (Watanabe et al., 2006). The artificial crossing experiments reported here revealed fruit sets from all possible combinations of different morphological taxa and different cpDNA phylogenetic subclades, exclusive of which taxon or cpDNA subclade was the ovule or pollen parent (Table 3). These results suggest that a prezygotic isolation barrier had not developed among either the morphological taxa or the cpDNA subclades, although the F1 hybrid fertility is indispensable for evaluating postzygotic isolating barrier.
Interestingly, when crossing direction (ovule or pollen parents) was considered, the frequencies of fruit sets were asymmetrical in two cases: (1) the cpDNA phylogenetic subclades A, BI, BII, BIII, or BIV as ovule parents and subclade BV as pollen parent, and (2) the morphological taxa Kaempferi or Tanzawana as ovule parents and Liukiuensis, Cucurbitifolia, or Mollissima as pollen parents (Table 3). The individuals used as the ovule parents came from the main islands of Japan, while individuals used as the pollen parents were derived from other areas, such as the Nansei Islands, Taiwan, and China (Fig. 1B). Following the tabulation of crossing data for the Japanese main islands vs. other areas, when the ovule parents were from the Japanese main islands and the pollen parents were from other areas, fruit set was markedly low (F/N = 7/130, 5.2%). Conversely, when the pollen parents were from the Japanese main islands and the ovule parents were from other areas, fruit set frequency was moderate (F/N = 36/78, 46.2%). Geologic data suggest that the land connection between the Japanese main islands and the Nansei Islands disappeared 1.3–1.7 million years ago (Kimura, 2002). Therefore, an asymmetric prezygotic isolating barrier, which was attributed to geological segregation and subsequent isolation, may have arisen between the populations of Japanese main islands and the populations of other areas represented by subclade BV.
Estimation of nuclear phylogeny
The five nuclear regions evaluated in this study had higher levels of polymorphism than did the combined cpDNA regions (Table 2), but resolution of the five phylogenetic trees was lower than that of cpDNA (Figs. 1, 3). The rooting of the five phylogenetic trees possess lower reliable, especially in AP3 exon and PI exon (Fig. 3). These likely reflect that diploid or polyploid nuclear genes have longer coalescence times than haploid cpDNA (Moore, 1995; Hare, 2001). The introns of AP3 and PI had more polymorphisms per sequence length than the exons in each gene (Table 2). Their polymorphisms were not associated with increased phylogenetic resolution (Fig. 3), perhaps due to convergent substitutions in the introns. These introns may be useful in detecting inter- or intraspecific variations to discriminate the A. kaempferi group populations, but may occasionally be misleading in reconstructing population histories.
The five phylogenetic trees had visually incongruent topologies, but show a similar pattern for a few individuals (Fig. 3). It is a matter of course that different nuclear genes on different chromosomes or genes effectively unlinked on the same chromosome have different patterns of divergence (Zhang and Hewitt, 2003; Avise, 2004). Even in the same locus, different regions (coding, noncoding introns, and noncoding flanking regions) may have different patterns of divergence as a result of differences in the level of selection (Zhang and Hewitt, 2003). The presence of discernable pattern between independent loci and/or different genomes suggests a more reliable estimate of population divergence (Hare, 2001; Sang, 2002; Zhang and Hewitt, 2003). Details of the similarity are discussed next.
Inconsistency and similarity among morphology, cpDNA, and nuclear DNA phylogenies
None of the five nuclear DNA phylogenetic trees for the 16 representative samples were congruent with either the morphological taxa or the cpDNA subclades in the A. kaempferi group, with the exception of a few interesting individuals (Fig. 3). The influences of incomplete lineage sorting, convergent evolution, and/or hybridization and introgression have been applied to explain inconsistency among morphological species and their gene trees (reviewed in Wendel and Doyle, 1998). However, previous studies revealed difficulty in assessing which evolutionary process was the most reasonable explanation (Brower et al., 1996; Rieseberg, 1997; Sang and Zhong, 2000; Holder et al., 2001). If artificial crossing between individuals from different morphological species is successful, then physiological reproductive barriers have not developed between species (Lowe et al., 2004), suggesting that natural hybridization and introgression events are one of the reasons for the inconsistency among morphological species and gene trees. From the results obtained in the present study, incomplete lineage sorting for cpDNA and/or nuclear DNA cannot be excluded as causing the inconsistency. Although morphological convergence is also expected from cpDNA phylogeny for Kaempferi in clades A, BI, BII, BIII, and BIV and for Shimadai in BI, BII, and BV (Fig. 1), we are unable to verify such an event from our nuclear data. However, our research demonstrated that the prezygotic isolating barrier was scarcely developed among the different morphological taxa and among the cpDNA subclades when crossing direction is not considered, which suggests the possibility that hybridization and introgression explains any inconsistency among morphology, cpDNA, and nuclear DNA.
On the other hand, the five nuclear DNA phylogenies have two commonalities (Fig. 3). One is that most nuclear DNA trees demonstrated distinct "Kaempferi with cpDNA clade A" samples, implying that "Kaempferi with clade A" populations may be differentiated from other populations. Another is that "Tanzawana with clade A" samples never formed a clade with "Kaempferi with clade A" samples, and these "Tanzawana with clade A" samples often shared an allele with the "Tanzawana with clade B" samples. The sharing of alleles means that chloroplast capture might have occurred between "Kaempferi with clade A" and "Tanzawana with clade B".
Subclade BV in cpDNA phylogeny was differentiated from other subclades by the asymmetric prezygotic isolation barrier, but it never formed a clade in any of the five nuclear DNA phylogenies (Fig. 3). The incongruence between cpDNA and nuclear DNA within subclade BV may be due to ancestral polymorphisms from incomplete lineage sorting for nuclear DNA than to hybridization and introgression between subclade BV and other geographically isolated subclades.
Significant correlation between cpDNA and the PI exon
To compare the phylogenetic and geographic patterns of variation in cpDNA and the PI exon, we extensively analyzed the PI exon (61 samples). The PI exon network clearly had two major lineages, and β, in the A. kaempferi group, and the two common haplotypes in each lineage (Fig. 4). The geographic distribution of the common haplotypes in each of the and β lineages showed a pattern quite similar to clades A and B in the cpDNA phylogeny, respectively (Fig. 5). This similarity, detected in different genomes, may reflect the evolutionary history of the A. kaempferi group. In a previous study using cpDNA (Watanabe et al., 2006), NCPA derived that past geographic fragmentations had resulted in two geographically differentiated clades: clade A in the southern Kanto region and clade B in the remainder of the A. kaempferi group range. With this scenario, the geographic distributions of the common PI exon haplotypes are likely to have paralleled the same past population fragmentation. As a result, the genealogies of clade A with the common haplotypes of the lineage, and of clade B with common haplotypes of the β lineage, may have been distributed in the southern Kanto region and the remainder of the A. kaempferi group range, respectively.
The notable point is that lineages and β of the PI exon did not completely correspond to clades A and B of the cpDNA phylogeny, respectively (Fig. 4). The rare haplotypes of the lineage were found from several individuals in cpDNA clade B, and four individuals in cpDNA clade A maintained the haplotypes of the β lineage (Fig. 5). Taking the crossing experiment results into account, this pattern indicates that pollen may have flowed between the two cpDNA clades. In the A. kaempferi group, pollen flow between populations is likely to be limited to a narrow geographic range because the tiny hump-backed flies of the family Phoridae, considered the major pollinator for the group (Iwata, 1975; Shiraiwa, 1991; K. Watanabe, personal observation), only fly short distances. This explanation verifies that the individuals of "the β haplotypes with clade A" and the individuals of "the common haplotypes with clade B" were restricted to the geographic boundary area delimited by two cpDNA clades in the southwest Kanto region (Fig. 5). The phylogeographic history based on cpDNA suggests that populations of the two major clades came into contact in the southwest Kanto region during the last postglacial range expansion (Watanabe et al., 2006). Here, pollen flow probably occurred across members of the two cpDNA clades in the recent past. On the other hand, the individuals of clade B with the rare haplotypes of the lineage were scattered in the western part of the range, far from the present contact zone of clades A and B. Previous work suggests that the A. kaempferi group has repeatedly experienced isolation and contact via contraction and expansion of populations, brought about by Quaternary climatic oscillations (Watanabe et al., 2006). In the lineage, each of the seven rare haplotypes has a few nucleotides that differ from the common haplotype, and the specific nucleotides that changed differ among the rare haplotypes. Because a certain amount of time was needed for these few nucleotide changes from the ancestral common haplotype to have occurred, multiple pollen transfers from clade A to B might have occurred through contact of the populations at different times in the distant past.
Speciation processes in the A. kaempferi group
The biological species concept defines species as reproductively isolated populations (e.g., Dobzhansky, 1937; Mayr, 1995; Mayden, 1997). However, in practice this definition is not readily applicable to all situations and can be difficult to apply to empirical data (Coyne and Orr, 2004) because speciation is a highly variable process, which differs in tempo and mode in different kinds of organisms (Avise, 2004).
The A. kaempferi group comprises six morphologically distinct taxa, but genetic variation and reproductive isolation data do not support the current morphological classification. A combination analysis of cpDNA and the PI exon revealed the existence of two major lineages, lacking prezygotic reproductive isolation between them but likely having experienced multiple hybridization and introgression. However, an asymmetric prezygotic isolating barrier occurred between populations in the Japanese main islands and in other geographic areas that belong to distinct cpDNA subclades. Therefore, it seems reasonable to conclude that development of prezygotic isolating mechanisms is not necessarily proportional to the degree of genetic divergence, and the biological species concept cannot be effectively applied to the A. kaempferi group. The evolutionary history of plants is more similar to a network than a cladogram for closely related species (Linder and Rieseberg, 2004). This network-like evolutionary history, sometimes called reticulate evolution, is characterized by occasional hybridization and subsequent fusion of different species. The deduced evolutionary history of the A. kaempferi group resembles reticulate evolution. From the result of previous NCPA using cpDNA (Watanabe et al., 2006), isolated glacial refugia preserving members of the A. kaempferi group must have promoted population differentiation, yet there has been insufficient time for complete speciation. During interglacial periods, populations expanded their ranges from separate glacial refugia, and their secondary contact might lead in part to gene exchange between differentiated populations. The gene flows brought by repeated contacts would be a major factor that has blurred the species boundaries.
Hybridization may cause rapid genetic variation, which is likely to promote adaptive evolution and speciation (Arnold, 1997; Seehausen, 2004). This study shows that multiple hybridization brought about by paleoclimatic oscillations is what contributed to creating plant diversity. The question remains whether the multiple hybridization events are related to the morphological diversification and subsequent speciation in the A. kaempferi group. It is notable that different morphological taxa are not sympatric and that few morphological intermediates are documented in the natural populations. Consequently, each morphological taxon may have already undergone some selection and drift, even though multiple hybridization events had created high levels of morphological variability. Future studies of artificial hybrid fertility and their corresponding floral morphology over several generations and an assessment of genetic identity using additional neutral and functional genes should contribute to clarifying these uncertainties.
FOOTNOTES
1 The authors thank Prof. C.-F. Hsieh, Dr. T. Fujita, and Mr. T. Siraiwa for their support of our fieldwork; Mr. K. Hirai and Mr. T. Ideno for cultivating samples; Dr. M. Sugiyama, Dr. M. Ohtani, Mr. K. Nagamiya, Dr. T. Kajita, and Dr. K. Takayama for technical advice; and Dr. H. Murata, Dr. T. Sugawara, Dr. N. Tanaka, and Ms. A. Maeda for providing samples. Funding for this project came from the Sasakawa Scientific Research Grant from The Japan Science Society (16-287K) to K.W. 
2 Current address: Musashi Junior & Senior High School, Tokyo 176-8535, Japan
3 Author for correspondence (e-mail: murata{at}ns.bg.s.u-tokyo.ac.jp)
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